316 Citations
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease a key virulence factor of the urinary tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry Here we report a crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment ... More
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme, urease; a key virulence factor of the urinary tract pathogen, Proteus mirabilis. We investigated the structural features of P. mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry. Here, we report a 2.0 Å crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer. Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment of putative nickel binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. Less
Human T-cell Leukemia Virus type HTLV- is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology especially when compared to the closely related virus HIV A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host Importantly the first in class anti-capsid drug approved lenacapavir can provide long-acting pre-exposure prophylaxis Such a property would provide a means to prevent the transmission of HTLV- but its capsid has not previously ... More
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. Less
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy StayGold a novel dimeric GFP recently monomerized through sequence engineering addresses this challenge with its high photostability There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets The unnatural amino acid -aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion Here we apply the same strategy to redshift StayGold through substitution of tyrosine- with -aminotyrosine The resultant red fluorescent protein StayRose shows an excitation wavelength maximum of nm and an ... More
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric GFP recently monomerized through sequence engineering, addresses this challenge with its high photostability. There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here, we apply the same strategy to redshift StayGold through substitution of tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favorable photostability in vivo in Escherichia coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step toward developing redshifted StayGold derivatives. Less
Heliorhodopsins HeRs the third rhodopsin family are characterized by inverted membrane topology and confinement to monoderm organisms yet their biological meaning has so far remained a mystery We report the first crystal structure of a eukaryotic HeR supported by structural modeling and comparative analyses across all domains of life A conserved carotenoid-binding site reminiscent of secondary antennae in some microbial rhodopsins is identified and found to be common among HeRs We show that inverted topology allows recruitment of exogenous xanthophylls inaccessible in diderm cells explaining HeRs distinctive orientation and distribution These findings reveal a previously unrecognized light-harvesting mechanism of HeRs ... More
Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, inaccessible in diderm cells, explaining HeRs’ distinctive orientation and distribution. These findings reveal a previously unrecognized light-harvesting mechanism of HeRs, expand the known repertoire of microbial phototrophy, and suggest evolutionary constraints linking membrane topology to environmental metabolite accessibility. Less
The composition of the primordial genetic material remains uncertain Studies of duplex structure and stability and of nonenzymatic template copying chemistry provide insight into the viability of potentially primordial genetic polymers Recent work suggests that - deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth Since DNA RNA duplexes are known to be less stable than RNA RNA duplexes we have examined the impact of dA dI and dG substitutions on RNA structure and nonenzymatic template copying We find that single -deoxyribo-purine substitutions reduce RNA duplex stability as expected Crystallographic studies show that such substitutions lead ... More
The composition of the primordial genetic material remains uncertain. Studies of duplex structure and stability, and of nonenzymatic template copying chemistry, provide insight into the viability of potentially primordial genetic polymers. Recent work suggests that 2′- deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth. Since DNA/RNA duplexes are known to be less stable than RNA/RNA duplexes, we have examined the impact of dA, dI, and dG substitutions on RNA structure and nonenzymatic template copying. We find that single 2′-deoxyribo-purine substitutions reduce RNA duplex stability, as expected. Crystallographic studies show that such substitutions lead to minimal structural changes but point to diminished solvation as a likely reason for duplex destabilization. Kinetic studies show that dI and dG substrates exhibit slightly weaker template binding and slower rates of template-directed primer extension than the corresponding ribo-purine substrates. In contrast, dA substrates exhibit much slower reaction kinetics but higher template affinity than rA substrates. Our results suggest that a mixed RNA/DNA primordial genetic polymer would have suffered from moderately slower rates of template copying, but that this could have been offset by an advantage due to more facile strand separation or exchange. Less
C domains are ubiquitous membrane-binding modules of residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification The genome of Trichomonas vaginalis the most common non-viral sexually transmitted human pathogen encodes eight genes that contain a N-terminal C module linked to a XYPPX-repeat domain of more than four XYPPX repeats C -XYPPX While the function of the XYPPX-repeat domain remains unknown its multiple association with C domains in T vaginalis suggests it is important The C domain from one of these C -XYPPX-repeat proteins Tv-C - was structurally and physically characterized using X-ray crystallography ... More
C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. The C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca2+ showed that Tv-C2-1 bound two Ca2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with Kds of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τc, in the apo (11.1 ns) and Ca2+-bound (9.2 ns) state suggests the protein becomes more compact upon Ca2+ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide 15N heteronuclear steady-state {1H}-15N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- continues to threaten global health This underpins the need for novel therapeutics against this virus Nonstructural protein Nsp of SARS-CoV- is a multifunctional protein with an essential role in viral replication As such it presents itself as an attractive target for drug discovery Here we describe two crystallographic fragment-screening campaigns against Nsp one using the established F X-Entry Screen and one using a new chemically and structurally diverse fragment library which we call the KIT library Together hits could be identified from screened fragments which constitutes the highest hit rate reported for Nsp to ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication. Less
Ferritins are a widespread family of proteins involved in iron homeostasis While classic ferritins consist of four -helices and form -meric nanocages related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity Here by analyzing the available genomic data we identify a family of double ferritin-like proteins DFLPs composed of two four-helical domains which arose by duplication of a ferritin fold protein We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens TmDFLP and CaDFLP and show that they form homodimers and bind heme We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase ... More
Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Less
In more and more drug discovery projects crystallographic fragment screening CFS is employed as an early screening method Here we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV- main protease While both crystal forms exhibit similar diffraction properties the observed hit rates in the two campaigns were vastly different For the monoclinic crystals a hit rate of was determined ... More
In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign. Less
Glutarimide analogs such as thalidomide redirect the E ubiquitin ligase CRL CRBN to induce degradation of certain zinc finger ZF proteins Although the core structural motif recognized by CRBN has been characterized it does not fully explain substrate specificity To explore the role of residues adjacent to this core motif we constructed a comprehensive ZF reporter library of reporters derived from human ZF proteins and conducted a library-on-library screen with glutarimide analogs to identify compounds that collectively degrade ZF reporters Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF ... More
Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design. Less
Transcription factors TFs regulate gene expression by engaging chromatin remodeling complexes yet the structural principles governing these critical interactions remain poorly defined Here we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU encoded by SPI directly engages the BAF mSWI SNF chromatin remodeling complex First using a variety of genomic approaches we establish that BAF collaborates with PU to regulate transcription in AML cells Then using a combination of biochemistry and biophysics mass spectrometry-based protein footprinting and crystallography we map the PU -BAF A interface to a disordered region of PU that adopts a helical conformation upon ... More
Transcription factors (TFs) regulate gene expression by engaging chromatin remodeling complexes, yet the structural principles governing these critical interactions remain poorly defined. Here, we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU.1 (encoded by SPI1) directly engages the BAF (mSWI/SNF) chromatin remodeling complex. First, using a variety of genomic approaches, we establish that BAF collaborates with PU.1 to regulate transcription in AML cells. Then, using a combination of biochemistry and biophysics, mass spectrometry-based protein footprinting, and crystallography, we map the PU.1-BAF60A interface to a disordered region of PU.1 that adopts a helical conformation upon binding to the YEATS-like domain of BAF60A. Disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. Finally, we conducted a high-throughput screen that yielded small molecules which selectively bind BAF60A and disrupt PU.1 binding. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a pioneer transcription factor and the BAF complex and establish a platform that enables targeting transcription factor-chromatin remodeling complex interactions in cancer. Less
Dual-specificity mitogen-activated protein kinase MAPK phosphatases MKPs directly dephosphorylate and inactivate the MAPKs Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored Here we sought to define the molecular mechanism of MKP regulation through an allosteric site within its catalytic domain We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y is required to maintain the structural integrity of the allosteric pocket Along with molecular dynamics simulations these data provide insight into how changes in ... More
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK Less
Transport and Golgi Organization Homolog TANGO protein deficiency disorder TDD is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay seizures intermittent ataxia hypothyroidism rhabdomyolysis life-threatening metabolic derangements and cardiac arrhythmias Mutations in TANGO result in mitochondrial dysfunction abnormal lipid homeostasis with cardiolipin deficiency and impaired Golgi-ER trafficking in TANGO patient-derived cells Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies the precise function of TANGO and the pathophysiology of TDD remain poorly understood A computationally derived three-dimensional structure model suggested that TANGO adopts an -fold similar to ... More
Transport and Golgi Organization 2 Homolog (TANGO2) protein deficiency disorder (TDD) is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay, seizures, intermittent ataxia, hypothyroidism, rhabdomyolysis, life-threatening metabolic derangements, and cardiac arrhythmias. Mutations in TANGO2 result in mitochondrial dysfunction, abnormal lipid homeostasis with cardiolipin deficiency, and impaired Golgi-ER trafficking in TANGO2 patient-derived cells. Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies, the precise function of TANGO2 and the pathophysiology of TDD remain poorly understood. A computationally derived three-dimensional structure model suggested that TANGO2 adopts an αββα-fold, similar to the N-terminal nucleophile aminohydrolase (Ntn) superfamily of proteins, but the experimentally verified structure has not been available thus far. Here, we present the first crystal structure of the recombinant human TANGO2, determined at 1.70 Å resolution. The X-ray structure data confirmed its predicted tertiary fold with similarity to the Ntn-hydrolase family of proteins, and the comparative analysis of the active site architecture, including residues involved in catalysis and putative ligand binding site, suggests a potential hydrolase function. Additional examination of the common mutation sites found in TDD patients provides insight regarding their potential effect on protein structure integrity. Less
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides climate-driven outbreaks and limited chemoprotective options Here we report the characterization of RUPB- the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase PfPKG RUPB- prevents infection by P falciparum and P cynomolgi sporozoites including the formation of hypnozoites by the latter A single oral dose blocks liver infection by P berghei sporozoites in vivo demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling The compound retains activity against field isolates resistant to chloroquine mefloquine cycloguanil sulfadoxine and pyrimethamine ... More
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided compound design and prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprotection and support further preclinical development of the RUPB-61 chemotype. Less
Malaria poses an enormous threat to human health With ever-increasing resistance to currently deployed antimalarials new targets and starting point compounds with novel mechanisms of action need to be identified Here we explore the antimalarial activity of the Streptomyces sp natural product -O-sulfamoyl- -chloroadenosine dealanylascamycin DACM and compare it with the synthetic adenosine monophosphate AMP mimic -O-sulfamoyladenosine AMS These nucleoside sulfamates exhibit potent inhibition of P falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin Exposure of P falciparum to DACM leads to inhibition of protein translation driven by eIF phosphorylation We show that DACM ... More
Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5ʹ-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition. Less
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions ... More
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death. Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects. Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown. We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy, and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF. Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions in five types of intermolecular high-affinity sites, leading to a decrease in negative surface charge and reversible oligomerization of the protein, thereby masking the target recognition sites responsible for its neurotrophic and antiangiogenic activities and collagen binding. Notably, PEDF secretion is enhanced in both glaucoma and retinal cell models in response to zinc stress; however, zinc binding negatively affects axogenic, differentiative and prosurvival functions of PEDF by suppressing its ability to activate receptor PEDF-R/PNPLA2. We suggest that glaucomatous neurodegeneration is associated with direct inhibition of PEDF signaling by extracellular zinc, making their complex a promising target for neuroprotective therapy. Less
Glycyl tRNA synthetases GlyRSs are prospective drug targets for combating Mycobacterium tuberculosis Mtb and cancer in humans These synthetases are of the -subtype with the ortholog in humans being dual targeted to the cytosol and mitochondria Whereas the human enzyme has been structurally characterized previously in several liganded states no structures of MtbGlyRS have thus far been reported Here we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS MtrGlyRS which progressed through all phases of the structural genomics pipeline for the purpose of facilitating structure-based drug discovery MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in ... More
Glycyl tRNA synthetases (GlyRSs) are prospective drug targets for combating Mycobacterium tuberculosis (Mtb) and cancer in humans. These synthetases are of the α2-subtype, with the ortholog in humans being dual targeted to the cytosol and mitochondria. Whereas the human enzyme has been structurally characterized previously in several liganded states, no structures of MtbGlyRS have thus far been reported. Here, we describe our recent work with MtbGlyRS and the closely-related Mycobacterium thermoresitibile GlyRS (MtrGlyRS), which progressed through all phases of the structural genomics pipeline, for the purpose of facilitating structure-based drug discovery. MtbGlyRS was expressed in Mycobacterium smegmatis and MtrGlyRS in Escherichia coli. Crystal structures are described for complexes of the two enzymes with adenosine monophosphate (AMP) and glycyl-sulfamoyl-adenylate (glycyl-AMS) at resolutions of 1.65/2.90 and 2.25/1.95 Å, respectively, and for MtrGlyRS in its apo state at 2.85 Å. Despite crystallizing in the dimeric state characteristic of many class II synthetases, the two enzymes elute predominantly as monomers during size exclusion chromatography. Strikingly, significant portions of the dimer interface and active site are unstructured in the MtrGlyRS apoenzyme crystal. AMP orders two tRNA recognition loops and a section of the insertion domain, and glycyl-AMS further stabilizes the structure, including the closure of a lid motif. Both the active and anticodon binding sites display structural differences with the human GlyRS and thus the collection of crystal structures should be useful for guiding drug development efforts targeting the various characterized structural states. Less
Room-temperature RT X-ray diffraction experiments enable us to investigate protein dynamics efficiently probe fragment binding and perform time-resolved crystallography experiments The Versatile Macromolecular Crystallography in-situ VMXi beamline at Diamond Light Source DLS in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals improving crystal integrity for fragile crystals While many X-ray sources are now equipped to grow crystals on site for in-situ experiments to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ... More
Room-temperature (RT) X-ray diffraction experiments enable us to investigate protein dynamics, efficiently probe fragment binding, and perform time-resolved crystallography experiments. The Versatile Macromolecular Crystallography in-situ (VMXi) beamline at Diamond Light Source (DLS) in the United Kingdom specializes in the collection of RT X-ray diffraction data in situ directly from crystallization trays without any manipulation of protein crystals, improving crystal integrity for fragile crystals. While many X-ray sources are now equipped to grow crystals on site for in-situ experiments, to date there has been no comprehensive analysis that we are aware of on the effect of shipping crystals on plates at ambient temperature for RT data collection, while the equivalent methodology for cryo-cooled crystals is well established. Here we examine the impact of shipping on crystals grown on MiTeGen In Situ-1 plates at the University of Buffalo Hauptman Woodward Research Institute (UB-HWI) in Buffalo, NY, United States transatlantic to DLS in Didcot, United Kingdom. We utilized the Stanford Synchrotron Radiation Lightsource (SSRL) Blue Box Thermal Shipper (Blue Box), which can maintain temperature for up to 168 hours, to ship crystallization plates at room temperature from UB-HWI to DLS. We hypothesized that long-distance shipping might compromise data quality through mechanical stress or temperature fluctuations. Instead, we found that room-temperature data collected at VMXi showed no significant differences for crystals set up at UB-HWI and shipped relative to crystals set up on site in the UK. High-resolution structures were successfully determined for all proteins in the study, demonstrating that long-distance shipment of crystals at non-cryogenic temperatures is feasible without compromising diffraction quality. This study provides a proof-of-concept workflow for expanding access to room-temperature crystallography worldwide, enabling more researchers to leverage cutting-edge techniques without needing to grow crystals on site. Less
In this work we present the high-resolution structure of human aconitate decarboxylase hACOD in its true apo form active site empty as well in complex with the inhibitor citraconate These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition Careful analysis of the structures indicates probable dynamics required for substrate inhibitor binding and catalysis These observations were further explored using molecular dynamic simulations which show a clear open-close mechanism of hACOD between the A and A loops the lid- and helical-domain respectively As part of the biochemical characterization of the protein we ... More
In this work, we present the high-resolution structure of human aconitate decarboxylase 1 (hACOD1) in its true apo form (active site empty) as well in complex with the inhibitor citraconate. These two new structures show the architecture of the active site and the structure-activity relationships of citraconate inhibition. Careful analysis of the structures indicates probable dynamics required for substrate/inhibitor binding and catalysis. These observations were further explored using molecular dynamic simulations, which show a clear open-close mechanism of hACOD1 between the A1 and A2 loops, the lid- and helical-domain respectively. As part of the biochemical characterization of the protein, we also developed an alternative kinetic assay which measures the rate of catalysis of hACOD1 by direct observation of the conversion of cis-aconitate to itaconate by NMR spectroscopy. The work herein offers a foundation for structure- and dynamic-driven design of novel hACOD1 inhibitors as novel chemotherapeutics. Less
Flavin-binding proteins flavoproteins are widespread in nature revealing versatile oxidation-reduction reactions and photochemistry Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics as well as fluorescent markers and photogenerators of reactive oxygen species Despite extensiev efforts all currently used LOV-derived proteins have similar absorption spectra with maxima around - and - nm Here we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein LSSFbFP which can be obtainedin vivo and in vitro with absorption maxima at - and - nm Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ... More
Flavin-binding proteins (flavoproteins) are widespread in nature, revealing versatile oxidation-reduction reactions and photochemistry. Flavoproteins derived from LOV domains are used for engineering of ligh-tresponsive tools in optogenetics, as well as fluorescent markers and photogenerators of reactive oxygen species. Despite extensiev efforts, all currently used LOV-derived proteins have similar absorption spectra with maxima around 275, 35-0375, and 450-485 nm. Here, we describe the discovery of a large Stokes shift flavi-nbased fluorescent protein, LSSFbFP, which can be obtainedin vivo and in vitro, with absorption maxima at 340-350 and 395-405 nm. Fluorescence emission of LSSFbFP mirrors that of classical FbFPs with the maximum at ~500 nm. We sho that the protein binds lumichrome as the chromophore and use low temperature and time-resolved spectroscopy, X-ray crystallography and modeling to prove that the apparent Stokes shift of LSSFbFP occurs due to excited state proton phenomena observed in flavoproteni s and pave the way for engineering of new flavin-based molecular instruments. Less
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae the essential extracellular cell-division-associated peptidoglycan PG hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX PcsB contains a cysteine hishdine-dependent amidohydrolase pephdase CHAP domain responsible for PG hydrolysis as well as a coiled-coil domain required for interaction with FtsEX ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane which ultimately regulates the PG hydrolase on the outside of the cell In this work we show using in vitro and in vivo approaches that the CHAP domain of PcsB predominately functions as ... More
In the human commensal Gram-positive bacterial pathogen Streptococcus pneumoniae, the essential extracellular cell-division-associated peptidoglycan (PG) hydrolase PcsB interacts directly with the cytoplasmic-membrane-bound complex between FtsE and FtsX (1–3). PcsB contains a cysteine, hishdine-dependent amidohydrolase/pephdase (CHAP) domain responsible for PG hydrolysis, as well as a coiled-coil domain required for interaction with FtsEX (1,4). ATP hydrolysis of FtsE in the cytoplasm drives conformational changes in FtsX in the cytoplasmic membrane, which ultimately regulates the PG hydrolase on the outside of the cell (5). In this work we show using in vitro and in vivo approaches, that the CHAP domain of PcsB predominately functions as an iso-D-Glutaminyl-Lysyl D,L-endopeptidase, with particular substrate specificity for Lys-containing, amidated PG, cleaving between the second and third amino acids of the peptidoglycan stem peptide. The catalytic activity of PcsB is regulated and activated by conformation changes of the coiled-coil region of PcsB and in part by a short helical region immediately adjacent to the CHAP domain to guard against PcsB hydrolytic activation outside of its cell division specific functional requirement. This work supports a model for the overall biological activity of the FtsEX-PcsB complex, in which ATP hydrolysis by FtsE in the cytoplasm, drives conformational changes in FtsX and PcsB resulting in the liberation of the hydrolytic CHAP domain of PcsB from its regulatory helix to allow PG stem peptide cleavage that splits the septal disk and marks a region of the peptidoglycan sacculus for subsequent cell division remodelling. Less
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life However chemical copying with the canonical nucleotides A U G and C strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U because of the stronger G C vs A U base pair and the weaker stacking interactions of U Recent advances in prebiotic chemistry suggest that the -thiopyrimidines were precursors to the canonical pyrimidines raising the possibility that they may have played an important early role in RNA copying chemistry Furthermore -thiouridine ... More
Nonenzymatic RNA copying is thought to have been responsible for the replication of genetic information during the origin of life. However, chemical copying with the canonical nucleotides (A, U, G, and C) strongly favors the incorporation of G and C and disfavors the incorporation of A and especially U, because of the stronger G:C vs. A:U base pair, and the weaker stacking interactions of U. Recent advances in prebiotic chemistry suggest that the 2-thiopyrimidines were precursors to the canonical pyrimidines, raising the possibility that they may have played an important early role in RNA copying chemistry. Furthermore, 2-thiouridine (s2U) and inosine (I) form by deamination of 2-thiocytidine (s2C) and A respectively. We used thermodynamic and crystallographic analyses to compare the I:s2C and A:s2U base pairs. We find that the I:s2C base pair is isomorphic and isoenergetic with the A:s2U base pair. The I:s2C base pair is weaker than a canonical G:C base pair, while the A:s2U base pair is stronger than the canonical A:U base pair, so that a genetic alphabet consisting of s2U, s2C, I and A generates RNA duplexes with uniform base pairing energies. Consistent with these results, kinetic analysis of nonenzymatic template-directed primer extension reactions reveals that s2C and s2U substrates bind similarly to I and A in the template, and vice versa. Our work supports the plausibility of a potentially primordial genetic alphabet consisting of s2U, s2C, I and A, and offers a potential solution to the long-standing problem of biased nucleotide incorporation during nonenzymatic template copying. Less
Immunomodulatory imide drugs IMiDs degrade specific C H zinc finger degrons in transcription factors making them effective against certain cancers SALL a cancer driver contains seven C H zinc fingers in four clusters including an IMiD degron in zinc finger cluster two ZFC Surprisingly IMiDs do not inhibit growth of SALL expressing cancer cells To overcome this limit we focused on a non-IMiD degron SALL zinc finger cluster four ZFC By combining AlphaFold and the ZFC -DNA crystal structure we identified a potential ZFC drug pocket Utilizing an in silico docking algorithm and cell viability assays we screened chemical libraries ... More
Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in four clusters, including an IMiD degron in zinc finger cluster two (ZFC2). Surprisingly, IMiDs do not inhibit growth of SALL4 expressing cancer cells. To overcome this limit, we focused on a non-IMiD degron, SALL4 zinc finger cluster four (ZFC4). By combining AlphaFold and the ZFC4-DNA crystal structure, we identified a potential ZFC4 drug pocket. Utilizing an in silico docking algorithm and cell viability assays, we screened chemical libraries and discovered SH6, which selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 led to significant 62% tumor growth inhibition of SALL4+ xenografts in vivo and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors in cancer. Less
Bacteriorhodopsin is a seven-helical light-driven proton pump and a model membrane protein Here we report engineering of soluble analogues of bacteriorhodopsin NeuroBRs which bind retinal and photocycle under illumination We also report the crystallographic structure of NeuroBR A determined at anisotropic resolution reaching that reveals a conserved chromophore binding pocket and tertiary structure Our results highlight the power of modern protein engineering approaches and pave the way towards wider development of molecular tools derived from membrane proteins
The Fe II - and -oxoglutarate -OG -dependent dioxygenase FOGDD enzyme superfamily catalyzes the oxidation of RNA DNA and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules For decades it has been appreciated that many FOGDDs require ascorbate Vitamin C as a cofactor for efficient catalysis but ascorbate requirements across different FOGDD enzymes its effects on the catalysis of different substrates and how it engages the FOGDD active site remain poorly understood Here we use RNA demethylases FTO and AlkBH as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions ... More
The Fe(II)- and 2-oxoglutarate(2-OG)-dependent dioxygenase (FOGDD) enzyme superfamily catalyzes the oxidation of RNA, DNA, and peptide substrates to install chemical modifications that regulate the diverse functions of these biomolecules. For decades, it has been appreciated that many FOGDDs require ascorbate (Vitamin C) as a cofactor for efficient catalysis, but ascorbate requirements across different FOGDD enzymes, its effects on the catalysis of different substrates, and how it engages the FOGDD active site, remain poorly understood. Here, we use RNA demethylases FTO and AlkBH5 as model FOGDD enzymes and show that their ascorbate requirements for efficient demethylation reactions are dramatically different. Furthermore, FTO was found to have strikingly different ascorbate requirements for its own demethylation reactions with different methylated RNA substrates. Our enzymology experiments suggest FTO and AlkBH5, and likely FOGDDs generally, can have widely different ascorbate dependencies based on the balance between an enzyme’s intrinsic ability to decarboxylate co-substrate 2-oxoglutarate and the kinetics of its substrate oxidation reaction. Finally, we determined a crystal structure of FTO in complex with ascorbate, which for the first time shows how ascorbate selectively engages the FOGDD active site. Together, our biochemical and structural data show that ascorbate can selectively tune the RNA demethylation reactions of FTO and AlkBH5, and that more globally, differences in ascorbate concentrations in different environments or diseases may regulate the activity and specificity of diverse FOGDD-catalyzed oxidation reactions in the cell. Less
Since the Macromolecular Crystallography MX group at Helmholtz-Zentrum Berlin HZB has been operating three MX beamlines at the BESSY II storage ring in Berlin These beamlines were established to support the emerging structural genomics initiatives founded in Germany Europe and overseas around the turn of the century Over the past two decades these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory a spectroscopy laboratory a Biosafety Level laboratory and all necessary computing resources for the MX and chemical crystallography user community Currently more than independent research groups ... More
Since 2003, the Macromolecular Crystallography (MX) group at Helmholtz-Zentrum Berlin (HZB) has been operating three MX beamlines at the BESSY II storage ring in Berlin. These beamlines were established to support the emerging structural genomics initiatives founded in Germany, Europe, and overseas around the turn of the century. Over the past two decades, these beamlines have been continuously developed to enable state-of-the-art diffraction experiments and to provide supporting facilities such as a sample preparation laboratory, a spectroscopy laboratory, a Biosafety Level 1 laboratory and all necessary computing resources for the MX and chemical crystallography user community. Currently, more than 100 independent research groups from the greater Berlin area, Germany, and Europe utilize these beamlines. Over time, more than 4500 Protein Data Bank depositions have been accrued based on data collected at the beamlines. This paper presents historical aspects of the beamlines, their current status including their research output, and future directions. Less
Bacterial proton pumps proteorhodopsins PRs are a major group of light-driven membrane proteins found in marine bacteria They are functionally and structurally distinct from archaeal and eukaryotic proton pumps To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level high-resolution structures of PRs functional states are needed In this work we have determined atomic-resolution structures of MAR a PR from marine actinobacteria in various functional states notably the challenging late O intermediate state These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward ... More
Bacterial proton pumps, proteorhodopsins (PRs), are a major group of light-driven membrane proteins found in marine bacteria. They are functionally and structurally distinct from archaeal and eukaryotic proton pumps. To elucidate the proton transfer mechanism by PRs and understand the differences to nonbacterial pumps on a molecular level, high-resolution structures of PRs’ functional states are needed. In this work, we have determined atomic-resolution structures of MAR, a PR from marine actinobacteria, in various functional states, notably the challenging late O intermediate state. These data and information from recent atomic-resolution structures on an archaeal outward proton pump bacteriorhodopsin and bacterial inward proton pump xenorhodopsin allow for deducing key universal elements for light-driven proton pumping. First, long hydrogen-bonded chains characterize proton pathways. Second, short hydrogen bonds allow proton storage and inhibit their backflow. Last, the retinal Schiff base is the active proton donor and acceptor to and from hydrogen-bonded chains. Less
NEMO is an essential component in the activation of the canonical NF B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO programmed to render this difficult protein domain amenable to NMR and X-ray characterization while preserving the biological function ZipNEMO binds IKK with nanomolar affinity is amenable ... More
NEMO is an essential component in the activation of the canonical NFκ B pathway and exerts its function by recruiting the I κ B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NFκ B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the design and characterization of novel engineered constructs of the IKK-binding domain of NEMO, programmed to render this difficult protein domain amenable to NMR and X-ray characterization, while preserving the biological function. ZipNEMO binds IKK β with nanomolar affinity, is amenable to heteronuclear NMR techniques and structure determination by X-ray crystallography. We show that NMR spectra of zipNEMO allow to detect inhibitor binding in solution and resonance assignment. The X-ray structure of zipNEMO highlights a novel ligand binding motif and the adaptability of the binding pocket and inspired the design of new peptide inhibitors. Less
Human lens fiber membrane intrinsic protein MP is the second most abundant membrane protein of the human eye lens Despite decades of effort its structure and function remained elusive Here we determined the MicroED structure of full-length human MP in lipidic-cubic phase to a resolution of MP forms tetramers each of which contain transmembrane -helices that are packed against one another forming a helical bundle Both the N- and C-termini of MP are cytoplasmic We found that each MP tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion These interactions were mediated by the extracellular loops of ... More
Human lens fiber membrane intrinsic protein MP20 is the second most abundant membrane protein of the human eye lens. Despite decades of effort its structure and function remained elusive. Here, we determined the MicroED structure of full-length human MP20 in lipidic-cubic phase to a resolution of 3.5 Å. MP20 forms tetramers each of which contain 4 transmembrane α-helices that are packed against one another forming a helical bundle. Both the N- and C-termini of MP20 are cytoplasmic. We found that each MP20 tetramer formed adhesive interactions with an opposing tetramer in a head-to-head fashion. These interactions were mediated by the extracellular loops of the protein. The dimensions of the MP20 adhesive junctions are consistent with the 11 nm thin lens junctions. Investigation of MP20 localization in human lenses indicated that in young fiber cells MP20 was stored intracellularly in vesicles and upon fiber cell maturation MP20 inserted into the plasma membrane and restricted the extracellular space. Together these results suggest that MP20 forms lens thin junctions in vivo confirming its role as a structural protein in the human eye lens, essential for its optical transparency. Less
Fatty acid esters of hydroxy fatty acids FAHFAs are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective large-scale production Enzymatic synthesis favored for its scalability and environmental impact is the preferred approach Candida Moesziomyces antarctica lipase A CalA previously known for its thermostability and limited ability to catalyze FAHFA esterification was investigated along with its orthologues for their ability to produce a variety of FAHFAs We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from ... More
Fatty acid esters of hydroxy fatty acids (FAHFAs) are a newly discovered lipid class known for their potential anti-inflammatory and insulin-sensitizing properties. A sustainable and efficient synthesis route is essential to realize the potential of FAHFAs and enable cost-effective, large-scale production. Enzymatic synthesis, favored for its scalability and environmental impact, is the preferred approach. Candida (Moesziomyces) antarctica lipase A (CalA), previously known for its thermostability and limited ability to catalyze FAHFA esterification, was investigated along with its orthologues for their ability to produce a variety of FAHFAs. We developed a systematic workflow to identify uncharacterized enzymes for FAHFA synthesis from natural sources, using an automation-compatible method, leading to the discovery of several novel lipases capable of synthesizing diverse FAHFAs. Among these lipases, two newly discovered enzymes, CL20 and CL23, demonstrated superior performance in FAHFA biosynthesis, achieving faster and higher yields than the benchmark enzyme, CalA. Our work advances methodologies and processes critical for industrial FAHFA production and provides a foundation for sustainable commercial-scale synthesis via synthetic enzymology. Less
Acetyl CoA synthetases ACS have emerged as drug targets for the treatment of cancer metabolic diseases as well as fungal and parasitic infections Although a variety of small molecule ACS inhibitors have been discovered the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition Through a chemical genetic-based synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C neoformans Acs relative to human ACSS as well as other fungal ACSs Xray crystallographic characterization of the ... More
Acetyl CoA synthetases (ACS) have emerged as drug targets for the treatment of cancer, metabolic diseases as well as fungal and parasitic infections. Although a variety of small molecule ACS inhibitors have been discovered, the systematic optimization of these molecules has been slowed by a lack of structural information regarding their mechanism of inhibition. Through a chemical genetic-based, synthetic lethal screen of the human fungal pathogen Cryptococcus neoformans, we identified an isoxazole-based ACS inhibitor with antifungal activity and exquisite selectivity for the C. neoformans Acs1 relative to human ACSS2 as well as other fungal ACSs. Xray crystallographic characterization of the isoxazole-CnAcs1 complex revealed that the isoxazole functions as an acetyl CoA mimic and occupies both the acetyl- and CoA-binding sites of CnAcs1. Consistent with this novel mode of inhibition, the isoxazoles display uncompetitive inhibition kinetics that are similar to antimalarial ACS inhibitors also proposed to target the CoA binding site. Consequently, these data provide structural and mechanistic insights into the remarkable selectivity of Acetyl CoA pocket-targeting ACS inhibitors. In addition, these data provide strong proof-of-principle that targeting fungal and parasitic ACSs for the development of novel anti-infectives can be achieved with high selectivity and, thereby, low host toxicity. Less
Crimean-Congo hemorrhagic fever virus CCHFV is a tickborne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we use structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-electron microscopy cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn ... More
Crimean-Congo hemorrhagic fever virus (CCHFV) is a tickborne virus that can cause severe disease in humans with case fatality rates of 10%–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we use structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-electron microscopy (cryo-EM) structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
The transcription factor BCL A is a critical regulator of the switch from fetal hemoglobin HbF to adult hemoglobin HbA during development BCL A binds at a cognate recognition site TGACCA in the -globin gene promoter and represses its expression DNA-binding is mediated by a triple zinc finger domain designated ZnF Here we report comprehensive investigation of ZnF leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA We delve into the dynamics and mode of interaction with DNA Moreover we discovered that the last zinc finger of BCL A ZnF plays a ... More
The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 β 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and β-thalassemia. Less
GPR is an orphan G protein coupled receptor with high constitutive activity found in D -type dopamine receptor expressing medium spiny neurons of the striatopallidal pathway which is aberrantly hyperactivated in Parkinson s disease Here we solved crystal structures of GPR without the addition of a ligand a pseudo-apo state and in complex with two inverse agonists including CVN which improved motor symptoms in patients with Parkinson s disease in clinical trials In addition we obtained a cryo electron microscopy structure of the signaling complex between GPR and its cognate Gs heterotrimer The pseudo-apo structure revealed a strong density in ... More
GPR6 is an orphan G protein–coupled receptor with high constitutive activity found in D2-type dopamine receptor–expressing medium spiny neurons of the striatopallidal pathway, which is aberrantly hyperactivated in Parkinson’s disease. Here, we solved crystal structures of GPR6 without the addition of a ligand (a pseudo-apo state) and in complex with two inverse agonists, including CVN424, which improved motor symptoms in patients with Parkinson’s disease in clinical trials. In addition, we obtained a cryo–electron microscopy structure of the signaling complex between GPR6 and its cognate Gs heterotrimer. The pseudo-apo structure revealed a strong density in the orthosteric pocket of GPR6 corresponding to a lipid-like endogenous ligand. A combination of site-directed mutagenesis, native mass spectrometry, and computer modeling suggested potential mechanisms for high constitutive activity and inverse agonism in GPR6 and identified a series of lipids and ions bound to the receptor. The structures and results obtained in this study could guide the rational design of drugs that modulate GPR6 signaling. Less
Heliorhodopsins HeRs constitute a novel and distinct group of microbial rhodopsins characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins The production of HeRs for structural and functional investigations has proven challenging as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date Notably no eukaryotic HeRs have been reported thus far In this study we report the first expression of three eukaryotic HeRs in the LEXSY expression system from marine and freshwater algae and a free-living marine unicellular eukaryote We spectroscopically characterized these ... More
Heliorhodopsins (HeRs) constitute a novel and distinct group of microbial rhodopsins, characterized by the inverted position of C- and N- termini relative to conventional Type I rhodopsins. The production of HeRs for structural and functional investigations has proven challenging, as evidenced by the structural elucidation of only two proteins and the functional characterization of a few others to date. Notably, no eukaryotic HeRs have been reported thus far. In this study, we report the first expression of three eukaryotic HeRs in the LEXSY expression system: from marine and freshwater algae and a free-living marine unicellular eukaryote. We spectroscopically characterized these HeRs, demonstrating that they were expressed in the functional states. Finally, we report their successful crystallization, thus paving the way for their further structural and functional studies Less
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have been found to sense only a single trigger during infection suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands Here we demonstrate that the antiphage defence protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using ... More
Eukaryotic innate immune systems use pattern recognition receptors to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators, known as bacteriophages1,2,3,4,5,6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have been found to sense only a single trigger during infection, suggesting a one-to-one relationship between bacterial pattern recognition receptors and their ligands7,8,9,10,11. Here we demonstrate that the antiphage defence protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the notable versatility of an immune sensory domain, which may be a common property of antiphage defence systems that enables them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbour both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phages can fully evade CapRelSJ46 defence only when both triggers are mutated. Our work shows how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow the detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defence systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less
Leishmania a protozoan parasite is responsible for significant morbidity and mortality worldwide manifesting as cutaneous mucocutaneous and visceral leishmaniasis These diseases pose a substantial burden especially in impoverished regions with limited access to effective medical treatments Current therapies are toxic have low efficacy and face growing resistance Understanding the metabolic pathways of Leishmania particularly those differing from its host can unveil potential therapeutic targets In this study we investigated the acetyl-CoA synthetase ACS enzyme from Leishmania infantum LiAcs which unlike many organisms also exhibits acetoacetyl-CoA synthetase KBC activity This dual functionality is unique among ANL superfamily enzymes and crucial for ... More
Leishmania, a protozoan parasite, is responsible for significant morbidity and mortality worldwide, manifesting as cutaneous, mucocutaneous, and visceral leishmaniasis. These diseases pose a substantial burden, especially in impoverished regions with limited access to effective medical treatments. Current therapies are toxic, have low efficacy, and face growing resistance. Understanding the metabolic pathways of Leishmania, particularly those differing from its host, can unveil potential therapeutic targets. In this study, we investigated the acetyl-CoA synthetase (ACS) enzyme from Leishmania infantum (LiAcs1), which, unlike many organisms, also exhibits acetoacetyl-CoA synthetase (KBC) activity. This dual functionality is unique among ANL superfamily enzymes and crucial for the parasite's reliance on leucine catabolism, energy production and sterol biosynthesis. Our biochemical characterization of LiAcs1 revealed its ability to utilize both acetate and acetoacetate substrates. Additionally, LiAcs1 displayed a distinct CoA substrate inhibition pattern, partially alleviated by acetoacetate. Structural analysis provided insights into the substrate binding flexibility of LiAcs1, highlighting a more promiscuous substrate pocket compared to other ACS or KBC-specific enzymes. Substrate mimetics elucidated its ability to accommodate both small and large AMP-ester derivatives, contributing to its dual ACS/KBC functionality. These findings not only advance our understanding of Leishmania metabolism but also present LiAcs1 as a promising drug target. The dual functionality of LiAcs1 underscores the potential for developing selective inhibitors that could disrupt critical metabolic pathways across Leishmania spp. as it appears this enzyme is highly conserved across this genus. This paves the way for developing novel effective treatments against this devastating disease. Less
Respiratory syncytial virus RSV causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age Vaccines based on the viral fusion protein are approved for adults over but infant protection relies on passive immunity via antibody transfer or maternal vaccination An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need Antibodies arising from the VH - VL - gene pairing can neutralize RSV without the need for affinity maturation making them attractive to target through vaccination Here we develop an anti-idiotypic monoclonal antibody ai-mAb immunogen that is specific for unmutated VH - ... More
Respiratory syncytial virus (RSV) causes lower respiratory tract infections with significant morbidity and mortality at the extremes of age. Vaccines based on the viral fusion protein are approved for adults over 60, but infant protection relies on passive immunity via antibody transfer or maternal vaccination. An infant vaccine that rapidly elicits protective antibodies would fulfill a critical unmet need. Antibodies arising from the VH3-21/VL1-40 gene pairing can neutralize RSV without the need for affinity maturation, making them attractive to target through vaccination. Here, we develop an anti-idiotypic monoclonal antibody (ai-mAb) immunogen that is specific for unmutated VH3-21/VL1-40 B cell receptors (BCRs). The ai-mAb efficiently engages B cells with bona fide target BCRs and does not activate off-target non-neutralizing B cells, unlike recombinant pre-fusion (preF) protein used in current RSV vaccines. These results establish proof of concept for using an ai-mAb-derived vaccine to target B cells hardwired to produce RSV-neutralizing antibodies. Less
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues F FAs in both street mixtures and counterfeit pills To expand current treatment options drug-targeting monoclonal antibodies mAbs offer a viable therapeutic for both pre- and postexposure clinical scenarios This study reports the isolation in vitro characterization and in vivo efficacy of two murine mAb families targeting fentanyl carfentanil or both Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil crystal structures of mAbs in complex with fentanyl or carfentanil were ... More
The opioid overdose epidemic is a growing and evolving public health crisis fueled by the widespread presence of fentanyl and fentanyl analogues (F/FAs) in both street mixtures and counterfeit pills. To expand current treatment options, drug-targeting monoclonal antibodies (mAbs) offer a viable therapeutic for both pre- and postexposure clinical scenarios. This study reports the isolation, in vitro characterization, and in vivo efficacy of two murine mAb families targeting fentanyl, carfentanil, or both. Because humanization of the mAbs by CDR grafting negatively impacted affinity for both fentanyl and carfentanil, crystal structures of mAbs in complex with fentanyl or carfentanil were analyzed to identify key residues involved in ligand binding in murine versus humanized structures, and site-directed mutagenesis was used to verify their functional importance. The structural analysis identified a framework residue, Tyr36, present in the murine germline sequence of two mAbs, which was critical for binding to fentanyl and carfentanil. These studies emphasize the importance of structural considerations in mAb engineering to optimize mAbs targeting small molecules including opioids and other drugs of public health interest. Less
Human - exonuclease PLD a member of the phospholipase D family of enzymes has been validated as a therapeutic target for treating Alzheimer's disease Here we have determined the crystal structure of the luminal domain of the enzyme at resolution revealing a bilobal structure with a catalytic site located between the lobes We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues previously shown to be key for phospholipase activity are not conserved or are absent This led us to test whether the enzyme ... More
Human 5′-3′ exonuclease PLD3, a member of the phospholipase D family of enzymes, has been validated as a therapeutic target for treating Alzheimer's disease. Here, we have determined the crystal structure of the luminal domain of the enzyme at 2.3 Å resolution, revealing a bilobal structure with a catalytic site located between the lobes. We then compared the structure with published crystal structures of other human PLD family members which revealed that a number of catalytic and lipid recognition residues, previously shown to be key for phospholipase activity, are not conserved or, are absent. This led us to test whether the enzyme is actually a phospholipase. We could not measure any phospholipase activity but the enzyme shows robust nuclease activity. Finally, we have mapped key single nucleotide polymorphisms onto the structure which reveals plausible reasons as to why they have an impact on Alzheimer's disease. Less
Photosensory receptors essential molecular entities across all domains of life enable organisms to detect and respond to light stimuli underpinning their critical involvement in regulating biological processes such as phototropism circadian rhythms photomorphogenesis and photosynthesis Among the myriad types of photosensory receptors blue light sensing proteins such as Blue Light Using Flavin BLUF photoreceptors distinguish themselves through their ability to utilize blue light for signalling Characterized by the conserved structure of their sensor domain BLUF photoreceptors are found in a wide array of organisms from bacteria and algae to plants and certain fungi Known for their capacity to bind flavin ... More
Photosensory receptors, essential molecular entities across all domains of life, enable organisms to
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less
detect and respond to light stimuli, underpinning their critical involvement in regulating biological
processes such as phototropism, circadian rhythms, photomorphogenesis, and photosynthesis.
Among the myriad types of photosensory receptors, blue light sensing proteins such as Blue Light
Using Flavin (BLUF) photoreceptors distinguish themselves through their ability to utilize blue
light for signalling. Characterized by the conserved structure of their sensor domain, BLUF
photoreceptors are found in a wide array of organisms, from bacteria and algae to plants and certain
fungi. Known for their capacity to bind flavin chromophores, typically flavin adenine dinucleotide
(FAD), they undergo conformational changes upon blue photon absorption, leading to downstream
signalling events, highlighting their pivotal role in the adaptive responses of various organisms to
light. This dissertation provides a comprehensive exploration of the BLUF photoreceptors,
particularly focusing on the Photoactivated Adenylate Cyclase protein from Oscillatoria acuminata
(OaPAC), which comprises a BLUF sensor domain linked to an Adenylate Cyclase (AC) effector
domain, catalysing the conversion of ATP into cAMP. This study aims to elucidate the
photoactivation mechanism of OaPAC and the ensuing signal transduction pathway, employing an
integrative approach that leverages time-resolved crystallography, small angle X-ray scattering,
spectroscopy, and biochemical characterization techniques. Special emphasis is placed on the TyrGln-Met triad in the BLUF domain, which plays a crucial role in the initial light-induced
rearrangements. Additionally, significant attention is given to the less understood aspects of BLUF
photoreceptors, particularly the transduction of the initial light signal to more distal parts of the
protein, which ultimately leads to biological activity. This research identifies a Metout/Trpin
transition as a crucial element in conveying the signal to the α-helix linker region. Finally, structural
models of OaPAC with ATP bound in the active site, along with complementary FTIR
investigations, provide a thorough understanding of ATP binding and allosteric communication. As
a result, the research presented in this dissertation not only expands the fundamental understanding
of BLUF photoreceptor biology, but also provides a framework for future studies aimed at
deciphering complete signal transduction pathways in multi-domain BLUF photoreceptors and
towards the development of optogenetic tools Less
VRC -class broadly neutralizing antibodies bnAbs have been isolated from people with HIV- but they have not yet been elicited by vaccination They are extensively somatically mutated and sometimes accumulate CDRL deletions Such indels may allow VRC -class antibodies to accommodate the glycans expressed on a conserved N N-linked glycosylation site in loop D of the gp subunit These glycans constitute a major obstacle in the development of VRC -class antibodies as unmutated antibody forms are unable to accommodate them Although immunizations of knock-in mice expressing human VRC -class B-cell receptors BCRs with specifically designed Env-derived immunogens lead to the ... More
VRC01-class broadly neutralizing antibodies (bnAbs) have been isolated from people with HIV-1, but they have not yet been elicited by vaccination. They are extensively somatically mutated and sometimes accumulate CDRL1 deletions. Such indels may allow VRC01-class antibodies to accommodate the glycans expressed on a conserved N276 N-linked glycosylation site in loop D of the gp120 subunit. These glycans constitute a major obstacle in the development of VRC01-class antibodies, as unmutated antibody forms are unable to accommodate them. Although immunizations of knock-in mice expressing human VRC01-class B-cell receptors (BCRs) with specifically designed Env-derived immunogens lead to the accumulation of somatic mutations in VRC01-class BCRs, CDRL1 deletions are rarely observed, and the elicited antibodies display narrow neutralizing activities. The lack of broad neutralizing potential could be due to the absence of deletions, the lack of appropriate somatic mutations, or both. To address this point, we modified our previously determined prime-boost immunization with a germline-targeting immunogen nanoparticle (426c.Mod.Core), followed by a heterologous core nanoparticle (HxB2.WT.Core), by adding a final boost with a cocktail of various stabilized soluble Env trimers. We isolated VRC01-like antibodies with extensive somatic mutations and, in one case, a seven-amino acid CDRL1 deletion. We generated chimeric antibodies that combine the vaccine-elicited somatic mutations with CDRL1 deletions present in human mature VRC01 bnAbs. We observed that CDRL1 indels did not improve the neutralizing antibody activities. Our study indicates that CDRL1 length by itself is not sufficient for the broadly neutralizing phenotype of this class of antibodies. Less
Regulation of Ras GTPases by GTPase activating proteins GAP is essential for their normal signaling Nine of the ten GAPs for Ras contain a C domain immediately proximal to their canonical GAP domain and in RasGAP p GAP p RasGAP RASA mutation of this domain is associated with vascular malformations in humans Here we show that the C domain of RasGAP is required for full catalytic activity towards Ras Analysis of the RasGAP C -GAP crystal structure AlphaFold models and sequence conservation reveal direct C domain interaction with the Ras allosteric lobe This is achieved by an evolutionarily conserved surface ... More
Regulation of Ras GTPases by GTPase activating proteins (GAP) is essential for their normal signaling. Nine of the ten GAPs for Ras contain a C2 domain immediately proximal to their canonical GAP domain, and in RasGAP (p120GAP, p120RasGAP; RASA1) mutation of this domain is associated with vascular malformations in humans. Here, we show that the C2 domain of RasGAP is required for full catalytic activity towards Ras. Analysis of the RasGAP C2-GAP crystal structure, AlphaFold models, and sequence conservation reveal direct C2 domain interaction with the Ras allosteric lobe. This is achieved by an evolutionarily conserved surface centered around RasGAP residue R707, point mutation of which impairs the catalytic advantage conferred by the C2 domain in vitro. In mice, R707C mutation phenocopies the vascular and signaling defects resulting from constitutive disruption of the RASA1 gene. In SynGAP, mutation of the equivalent conserved C2 domain surface impairs catalytic activity. Our results indicate that the C2 domain is required to achieve full catalytic activity of Ras GTPase activating proteins. Less
Bacillus circulans xylanase BcX from the glycoside hydrolase family degrades xylan through a retaining double-displacement mechanism The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large active site cleft with six subsites allowing the binding of six xylose units Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways In the crystal structure of the catalytically inactive variant BcX E Q the substrate xylotriose is observed in the active site as well as bound to the known secondary binding site and a third site on the protein surface Nuclear ... More
Bacillus circulans xylanase (BcX) from the glycoside hydrolase family 11 degrades xylan through a retaining, double-displacement mechanism. The enzyme is thought to hydrolyze glycosidic bonds in a processive manner and has a large, active site cleft, with six subsites allowing the binding of six xylose units. Such an active site architecture suggests that oligomeric xylose substrates can bind in multiple ways. In the crystal structure of the catalytically inactive variant BcX E78Q, the substrate xylotriose is observed in the active site, as well as bound to the known secondary binding site and a third site on the protein surface. Nuclear magnetic resonance (NMR) titrations with xylose oligomers of different lengths yield nonlinear chemical shift trajectories for active site nuclei resonances, indicative of multiple binding orientations for these substrates for which binding and dissociation are in fast exchange on the NMR timescale, exchanging on the micro- to millisecond timescale. Active site binding can be modeled with a 2 : 1 model with dissociation constants in the low and high millimolar range. Extensive mutagenesis of active site residues indicates that tight binding occurs in the glycon binding site and is stabilized by Trp9 and the thumb region. Mutations F125A and W71A lead to large structural rearrangements. Binding at the glycon site is sensed throughout the active site, whereas the weak binding mostly affects the aglycon site. The interactions with the two active site locations are largely independent of each other and of binding at the secondary binding site. Less
In diderm bacteria the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane IM to the outer membrane OM and therefore plays an essential role in bacterial envelope homeostasis After extrusion of modified lipoproteins from the IM via the LolCDE complex the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB itself a lipoprotein with a LolA-like fold Yet LolB homologs appear restricted to -proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi suggesting a different hand-off mechanism at the OM Here we ... More
In diderm bacteria, the Lol pathway canonically mediates the periplasmic transport of lipoproteins from the inner membrane (IM) to the outer membrane (OM) and therefore plays an essential role in bacterial envelope homeostasis. After extrusion of modified lipoproteins from the IM via the LolCDE complex, the periplasmic chaperone LolA carries lipoproteins through the periplasm and transfers them to the OM lipoprotein insertase LolB, itself a lipoprotein with a LolA-like fold. Yet, LolB homologs appear restricted to ψ-proteobacteria and are missing from spirochetes like the tick-borne Lyme disease pathogen Borrelia burgdorferi, suggesting a different hand-off mechanism at the OM. Here, we solved the crystal structure of the B. burgdorferi LolA homolog BB0346 (LolABb) at 1.9 Å resolution. We identified multiple structural deviations in comparative analyses to other solved LolA structures, particularly a unique LolB-like protruding loop domain. LolABb failed to complement an Escherichia coli lolA knockout, even after codon optimization, signal I peptide adaptation, and a C-terminal chimerization which had allowed for complementation with an α-proteobacterial LolA. Analysis of a conditional B. burgdorferi lolA knockout strain indicated that LolABb was essential for growth. Intriguingly, protein localization assays indicated that initial depletion of LolABb led to an emerging mislocalization of both IM and periplasmic OM lipoproteins, but not surface lipoproteins. Together, these findings further support the presence of two separate primary secretion pathways for periplasmic and surface OM lipoproteins in B. burgdorferi and suggest that the distinct structural features of LolABb allow it to function in a unique LolB-deficient lipoprotein sorting system. Less
Iron storage proteins e g vertebrate ferritin and the ferritin-like bacterioferritin Bfr and bacterial ferritin Ftn are spherical hollow proteins that catalyze the oxidation of Fe at binuclear iron ferroxidase centers FOC and store the Fe in their interior thus protecting cells from unwanted Fe Fe redox cycling and storing iron at concentrations far above the solubility of Fe Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC Bfr and Ftn were thought to coexist in bacteria as homopolymers but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits ... More
Iron storage proteins, e.g., vertebrate ferritin, and the ferritin-like bacterioferritin (Bfr) and bacterial ferritin (Ftn), are spherical, hollow proteins that catalyze the oxidation of Fe2+ at binuclear iron ferroxidase centers (FOC) and store the Fe3+ in their interior, thus protecting cells from unwanted Fe3+/Fe2+ redox cycling and storing iron at concentrations far above the solubility of Fe3+. Vertebrate ferritins are heteropolymers of H and L subunits with only the H subunits having FOC. Bfr and Ftn were thought to coexist in bacteria as homopolymers, but recent evidence indicates these molecules are heteropolymers assembled from Bfr and Ftn subunits. Despite the heteropolymeric nature of vertebrate and bacterial ferritins, structures have been determined only for recombinant proteins constituted by a single subunit type. Herein we report the structure of Acinetobacter baumannii bacterioferritin, the first structural example of a heteropolymeric ferritin or ferritin-like molecule, assembled from completely overlapping Ftn homodimers harboring FOC and Bfr homodimers devoid of FOC but binding heme. The Ftn homodimers function by catalyzing the oxidation of Fe2+ to Fe3+, while the Bfr homodimers bind a cognate ferredoxin (Bfd) which reduces the stored Fe3+ by transferring electrons via the heme, enabling Fe2+ mobilization to the cytosol for incorporation in metabolism. Less
Inhibition of the proteolytic processing of hepatocyte growth factor HGF and macrophage stimulating protein MSP is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis Here we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries PS-SCL technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF MSP activating serine proteases HGFA matriptase and hepsin These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease We demonstrate that by varying the P P and P positions of the inhibitor ... More
Inhibition of the proteolytic processing of hepatocyte growth factor (HGF) and macrophage stimulating protein (MSP) is an attractive approach for the drug discovery of novel anticancer therapeutics which prevent tumor progression and metastasis. Here, we utilized an improved and expanded version of positional scanning of substrate combinatorial libraries (PS-SCL) technique called HyCoSuL to optimize peptidomimetic inhibitors of the HGF/MSP activating serine proteases, HGFA, matriptase, and hepsin. These inhibitors have an electrophilic ketone serine trapping warhead and thus form a reversible covalent bond to the protease. We demonstrate that by varying the P2, P3, and P4 positions of the inhibitor with unnatural amino acids based on the protease substrate preferences learned from HyCoSuL, we can predictably modify the potency and selectivity of the inhibitor. We identified the tetrapeptide JH-1144 (8) as a single digit nM inhibitor of HGFA, matriptase and hepsin with excellent selectivity over Factor Xa and thrombin. These unnatural peptides have increased metabolic stability relative to natural peptides of similar structure. The tripeptide inhibitor PK-1-89 (2) has excellent pharmacokinetics in mice with good compound exposure out to 24 h. In addition, we obtained an X-ray structure of the inhibitor MM1132 (15) bound to matriptase revealing an interesting binding conformation useful for future inhibitor design. Less
Nontuberculous mycobacteria NTM are emerging human pathogens linked to severe pulmonary diseases Current treatments involve the prolonged use of multiple drugs and are often ineffective Bacterial dihydrofolate reductase DHFR is a key enzyme targeted by antibiotics in Gram-negative bacterial infections However existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs Here we detail the rational design of NTM DHFR inhibitors based on P a malarial DHFR inhibitor We identified a -diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species Mycobacterium avium and Mycobacterium abscessus This study underscores ... More
Nontuberculous mycobacteria (NTM) are emerging human pathogens linked to severe pulmonary diseases. Current treatments involve the prolonged use of multiple drugs and are often ineffective. Bacterial dihydrofolate reductase (DHFR) is a key enzyme targeted by antibiotics in Gram-negative bacterial infections. However, existing DHFR inhibitors designed for Gram-negative bacteria often fail against mycobacterial DHFRs. Here, we detail the rational design of NTM DHFR inhibitors based on P218, a malarial DHFR inhibitor. We identified 8, a 2,4-diaminopyrimidine exhibiting improved pharmacological properties and activity against purified DHFR and whole cell cultures of two predominant NTM species: Mycobacterium avium and Mycobacterium abscessus. This study underscores the potential of 8 as a promising candidate for the in vivo validation of DHFR as an effective treatment against NTM infections. Less
In cyanobacteria Elongation factor Tu EF-Tu plays a crucial role in the repair of photosystem II PSII which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species ROS However the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear Previous research has shown that a mutated EF-Tu where C is substituted with a Ser residue can alleviate photoinhibition highlighting the important role of C in EF-Tu photosensitivity In this study we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form ... More
In cyanobacteria, Elongation factor Tu (EF-Tu) plays a crucial role in the repair of photosystem II (PSII), which is highly susceptible to oxidative stress induced by light exposure and regulated by reactive oxygen species (ROS). However, the specific molecular mechanism governing the functional regulation of EF-Tu by ROS remains unclear. Previous research has shown that a mutated EF-Tu, where C82 is substituted with a Ser residue, can alleviate photoinhibition, highlighting the important role of C82 in EF-Tu photosensitivity. In this study, we elucidated how ROS deactivate EF-Tu by examining the crystal structures of EF-Tu in both wild-type and mutated form (C82S) individually at resolutions of 1.7 Å and 2.0 Å in Synechococcus elongatus PCC 7942 complexed with GDP. Specifically, the GDP-bound form of EF-Tu adopts an open conformation with C82 located internally, making it resistant to oxidation. Coordinated conformational changes in switches I and II create a tunnel that positions C82 for ROS interaction, revealing the vulnerability of the closed conformation of EF-Tu to oxidation. An analysis of these two structures reveals that the precise spatial arrangement of C82 plays a crucial role in modulating EF-Tu's response to ROS, serving as a regulatory element that governs photosynthetic biosynthesis. Less
Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes Deubiquitinases DUBs are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases including cancer Ubiquitin specific proteases USPs which constitute the largest family of DUBs in humans have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment Two related members ... More
Ubiquitination is an important post-translational modification that maintains cellular homeostasis by regulating various biological processes. Deubiquitinases (DUBs) are enzymes that reverse the ubiquitination process by catalyzing the removal of ubiquitin from a substrate. Abnormal expression or function of DUBs is often associated with the onset and progression of various diseases, including cancer. Ubiquitin specific proteases (USPs), which constitute the largest family of DUBs in humans, have become the center of interest as potential targets in cancer therapy as many of them display increased activity or are overexpressed in a range of malignant tumors or the tumor microenvironment. Two related members of the USP family, USP28 and USP25, share high sequence identities but play diverse biological roles. USP28 regulates cell proliferation, oncogenesis, DNA damage repair and apoptosis, whereas USP25 is involved in the anti-viral response, innate immunity and ER-associated degradation in addition to carcinogenesis. USP28 and USP25 also exhibit different oligomeric states – while USP28 is a constitutively active dimer, USP25 assumes an auto-inhibited tetrameric structure. The catalytic domains of both USP28 and USP25 comprise the canonical, globular USP-domain but contain an additional, extended insertion site called USP25/28 catalytic domain inserted domain (UCID) that mediates oligomerization of the proteins. Disruption of the USP25 tetramer leads to the formation of an activated dimeric protein. However, it is still not clear what triggers its activation. Due to their role in maintaining and stabilizing numerous oncoproteins, USP28 and USP25 have emerged as interesting candidates for anti-cancer therapy. Recent advances in smallmolecular inhibitor development have led to the discovery of relatively potent inhibitors of USP28 and USP25. This thesis focuses on the structural elucidation of USP28 and the biochemical characterization of USP28/USP25, both in complex with representatives of three out of the eight compound classes reported as USP28/USP25-specific inhibitors. The crystal structures of USP28 in complex with the AZ compounds, Vismodegib and FT206 reveal that all three inhibitor classes bind into the same allosteric pocket distant from the catalytic center, located between the palm and the thumb subdomains (the S1-site). Intriguingly, this binding pocket is identical to the UCID-tip binding interface in the USP25 tetramer, rendering the protein in a locked, inactive conformation. Formation of the binding pocket in USP28 requires a shift in the helix α5, which induces conformational changes and local distortion of the binding channel that typically accommodates the C-terminal tail of I Ubiquitin, thus preventing catalysis and abrogating USP28 activity. The key residues of the USP28-inhibitor binding pocket are highly conserved in USP25. Mutagenesis studies of these residues accompanied by biochemical and biophysical assays confirm the proposed mechanism of inhibition and similar binding to USP25. This work provides valuable insights into the inhibition mechanism of the small molecule compounds specifically for the DUBs USP28 and USP25. The USP28-inhibitor complex structures offer a framework to develop more specific and potent inhibitors. Less
Zoonotic coronaviruses are known to produce severe infections in humans and have been the cause of significant morbidity and mortality worldwide SARS-CoV- was the largest and latest contributor of fatal cases even though MERS-CoV has the highest case-fatality ratio among zoonotic coronaviruses These infections pose a high risk to public health worldwide warranting efforts for the expeditious discovery of antivirals Hence we hereby describe a novel series of inhibitors of coronavirus CLpro embodying an N-substituted -pyrrolidone scaffold envisaged to exploit favorable interactions with the S S subsites and connected to an invariant Leu-Gln P P recognition element Several inhibitors showed ... More
Zoonotic coronaviruses are known to produce
severe infections in humans and have been the cause of significant
morbidity and mortality worldwide. SARS-CoV-2 was the largest
and latest contributor of fatal cases, even though MERS-CoV has
the highest case-fatality ratio among zoonotic coronaviruses. These
infections pose a high risk to public health worldwide warranting
efforts for the expeditious discovery of antivirals. Hence, we hereby
describe a novel series of inhibitors of coronavirus 3CLpro
embodying an N-substituted 2-pyrrolidone scaffold envisaged to
exploit favorable interactions with the S3−S4 subsites and
connected to an invariant Leu-Gln P2−P1 recognition element.
Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution
crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants
associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the
inhibitors. Less
severe infections in humans and have been the cause of significant
morbidity and mortality worldwide. SARS-CoV-2 was the largest
and latest contributor of fatal cases, even though MERS-CoV has
the highest case-fatality ratio among zoonotic coronaviruses. These
infections pose a high risk to public health worldwide warranting
efforts for the expeditious discovery of antivirals. Hence, we hereby
describe a novel series of inhibitors of coronavirus 3CLpro
embodying an N-substituted 2-pyrrolidone scaffold envisaged to
exploit favorable interactions with the S3−S4 subsites and
connected to an invariant Leu-Gln P2−P1 recognition element.
Several inhibitors showed nanomolar antiviral activity in enzyme and cell-based assays, with no significant cytotoxicity. Highresolution
crystal structures of inhibitors bound to the 3CLpro were determined to probe and identify the molecular determinants
associated with binding, to inform the structure-guided optimization of the inhibitors, and to confirm the mechanism of action of the
inhibitors. Less
Lactate dehydrogenase-A LDHA is the major isoform of lactate dehydrogenases LDH that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma PDAC Despite some progress current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif Compounds and are structurally related analogs that demonstrated potent inhibition of LDHA with IC s of nM and nM respectively We solved cocrystal structures of compound -bound to LDHA that showed that the ... More
Lactate dehydrogenase-A (LDHA) is the major isoform of lactate dehydrogenases (LDH) that is overexpressed and linked to poor survival in pancreatic ductal adenocarcinoma (PDAC). Despite some progress, current LDH inhibitors have poor structural and physicochemical properties or exhibit unfavorable pharmacokinetics that have hampered their development. The present study reports the synthesis and biological evaluation of a novel class of LDHA inhibitors comprising a succinic acid monoamide motif. Compounds 6 and 21 are structurally related analogs that demonstrated potent inhibition of LDHA with IC50s of 46 nM and 72 nM, respectively. We solved cocrystal structures of compound 21-bound to LDHA that showed that the compound binds to a distinct allosteric site between the two subunits of the LDHA tetramer. Inhibition of LDHA correlated with reduced lactate production and reduction of glycolysis in MIA PaCa-2 pancreatic cancer cells. The lead compounds inhibit the proliferation of human pancreatic cancer cell lines and patient-derived 3D organoids and exhibit a synergistic cytotoxic effect with the OXPHOS inhibitor phenformin. Unlike current LDHA inhibitors, 6 and 21 have appropriate pharmacokinetics and ligand efficiency metrics, exhibit up to 73% oral bioavailability, and a cumulative half-life greater than 4 h in mice. Less
We report the biochemical structural and functional characterization of the protein coded by gene PA in the P aeruginosa PAO genome The PA gene had been annotated as coding a probable bacterioferritin Our structural work shows that the product of gene PA is a protein that adopts the Dps subunit fold which oligomerizes into a -mer quaternary structure Unlike Dps however the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit in a manner identical to that observed in the ferroxidase center of P aeruginosa bacterioferritin Since these structural characteristics correspond to Dps-like proteins we term the ... More
We report the biochemical, structural, and functional characterization of the protein coded by gene PA4880 in the P. aeruginosa PAO1 genome. The PA4880 gene had been annotated as coding a probable bacterioferritin. Our structural work shows that the product of gene PA4880 is a protein that adopts the Dps subunit fold, which oligomerizes into a 12-mer quaternary structure. Unlike Dps, however, the ferroxidase di-iron centers and iron coordinating ligands are buried within each subunit, in a manner identical to that observed in the ferroxidase center of P. aeruginosa bacterioferritin. Since these structural characteristics correspond to Dps-like proteins, we term the protein as P. aeruginosa Dps-like, or Pa DpsL. The ferroxidase centers in Pa DpsL catalyze the oxidation of Fe2+ utilizing O2 or H2O2 as oxidant, and the resultant Fe3+ is compartmentalized in the interior cavity. Interestingly, incubating Pa DpsL with plasmid DNA results in efficient nicking of the DNA and at higher concentrations of Pa DpsL the DNA is linearized and eventually degraded. The nickase and endonuclease activities suggest that Pa DpsL, in addition to participating in the defense of P. aeruginosa cells against iron-induced toxicity, may also participate in the innate immune mechanisms consisting of restriction endonucleases and cognate methyl transferases. Less
Given the crucial role of the main protease Mpro in the replication cycle of SARS-CoV- this viral cysteine protease constitutes a high-profile drug target We investigated peptidomimetic azapeptide nitriles as auspicious irreversibly acting inhibitors of Mpro Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design Encouraged by the bioactive conformation of open-chain inhibitors we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor with an IC value of ... More
Given the crucial role of the main protease (Mpro) in the replication cycle of SARS-CoV-2, this viral cysteine protease constitutes a high-profile drug target. We investigated peptidomimetic azapeptide nitriles as auspicious, irreversibly acting inhibitors of Mpro. Our systematic approach combined an Mpro active-site scanning by combinatorially assembled azanitriles with structure-based design. Encouraged by the bioactive conformation of open-chain inhibitors, we conceptualized the novel chemotype of macrocyclic azanitriles whose binding mode was elucidated by cocrystallization. This strategy provided a favorable entropic contribution to target binding and resulted in the development of the extraordinarily potent Mpro inhibitor 84 with an IC50 value of 3.23 nM and a second-order rate constant of inactivation, kinac/Ki, of 448,000 M–1s–1. The open-chain Mpro inhibitor 58, along with the macrocyclic compounds 83 and 84, a broad-spectrum anticoronaviral agent, demonstrated the highest antiviral activity with EC50 values in the single-digit micromolar range. Our findings are expected to promote the future development of peptidomimetic Mpro inhibitors as anti-SARS-CoV-2 agents. Less
Effector proteins are central to the success of plant pathogens while immunity in hostplants is driven by receptor-mediated recognition of these effectors Understanding the mole-cular details of effector receptor interactions is key for the engineering of novel immunereceptors Here we experimentally determined the crystal structure of the Puccinia graminis f sp tri-tici Pgt effector AvrSr which was not accurately predicted using AlphaFold We charac-terised the role of the conserved cysteine residues in AvrSr using in vitro biochemical assaysand examined Sr -mediated recognition using transient expression in Nicotiana spp andwheat protoplasts The AvrSr structure contains a novel b-strand rich modular fold ... More
Effector proteins are central to the success of plant pathogens, while immunity in hostplants is driven by receptor-mediated recognition of these effectors. Understanding the mole-cular details of effector–receptor interactions is key for the engineering of novel immunereceptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tri-tici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We charac-terised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assaysand examined Sr27-mediated recognition using transient expression in Nicotiana spp. andwheat protoplasts. The AvrSr27 structure contains a novel b-strand rich modular fold consisting of two structu-rally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient forinteraction with Sr27 and triggering cell death. We identified two Pgt proteins structurallyrelated to AvrSr27 but with low sequence identity that can also associate with Sr27, albeitmore weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in tran-sient expression systems. Collectively, our findings have important implications for utilising protein prediction plat-forms for effector proteins, and those embarking on bespoke engineering of immunity recep-tors as solutions to plant disease Less
Cyanobacterial CO concentrating mechanisms CCMs sequester a globally consequential proportion of carbon into the biosphere Proteinaceous microcompartments called carboxysomes play a critical role in CCM function housing two enzymes to enhance CO fixation carbonic anhydrase CA and Rubisco Despite its importance our current understanding of the carboxysomal CAs found in -cyanobacteria CsoSCA remains limited particularly regarding the regulation of its activity Here we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp PCC Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose- -bisphosphate and forms a hexameric trimer of dimers ... More
Cyanobacterial CO2 concentrating mechanisms (CCMs) sequester a globally consequential proportion of carbon into the biosphere. Proteinaceous microcompartments, called carboxysomes, play a critical role in CCM function, housing two enzymes to enhance CO2 fixation: carbonic anhydrase (CA) and Rubisco. Despite its importance, our current understanding of the carboxysomal CAs found in α-cyanobacteria, CsoSCA, remains limited, particularly regarding the regulation of its activity. Here, we present a structural and biochemical study of CsoSCA from the cyanobacterium Cyanobium sp. PCC7001. Our results show that the Cyanobium CsoSCA is allosterically activated by the Rubisco substrate ribulose-1,5-bisphosphate and forms a hexameric trimer of dimers. Comprehensive phylogenetic and mutational analyses are consistent with this regulation appearing exclusively in cyanobacterial α-carboxysome CAs. These findings clarify the biologically relevant oligomeric state of α-carboxysomal CAs and advance our understanding of the regulation of photosynthesis in this globally dominant lineage. Less
Eukaryotic innate immune systems use pattern recognition receptors PRRs to sense infection by detecting pathogen-associated molecular patterns which then triggers an immune response Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages Although different immunity proteins can recognize different phage-encoded triggers individual bacterial immunity proteins have only been found to sense a single trigger during infection suggesting a one-to-one relationship between bacterial PRRs and their ligands Here we demonstrate that the anti-phage defense protein CapRelSJ in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the ... More
Eukaryotic innate immune systems use pattern recognition receptors (PRRs) to sense infection by detecting pathogen-associated molecular patterns, which then triggers an immune response. Bacteria have similarly evolved immunity proteins that sense certain components of their viral predators known as bacteriophages1–6. Although different immunity proteins can recognize different phage-encoded triggers, individual bacterial immunity proteins have only been found to sense a single trigger during infection, suggesting a one-to-one relationship between bacterial PRRs and their ligands7–11. Here, we demonstrate that the anti-phage defense protein CapRelSJ46 in Escherichia coli can directly bind and sense two completely unrelated and structurally different proteins using the same sensory domain, with overlapping but distinct interfaces. Our results highlight the remarkable versatility of an immune sensory domain, which may be a common property of anti-phage defense systems and enable them to keep pace with their rapidly evolving viral predators. We found that Bas11 phages harbor both trigger proteins that are sensed by CapRelSJ46 during infection, and we demonstrate that such phage can only fully evade CapRelSJ46 defense when both triggers are mutated. Our work reveals how a bacterial immune system that senses more than one trigger can help prevent phages from easily escaping detection, and it may allow detection of a broader range of phages. More generally, our findings illustrate unexpected multifactorial sensing by bacterial defense systems and complex coevolutionary relationships between them and their phage-encoded triggers. Less
The adenosine A A receptor A AAR belongs to the rhodopsin-like G protein-coupled receptor GPCR family which constitutes the largest class of GPCRs Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist Here we determined an X-ray crystal structure of the partial A AAR agonist -amino- - H-imidazol- -ylmethyl sulfanyl - -p-hydroxyphenyl- -pyridinedicarbonitrile LUF in complex with the A AAR construct A A-PSB -bRIL stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket The determined high-resolution structure resolved water ... More
The adenosine A2A receptor (A2AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family, which constitutes the largest class of GPCRs. Partial agonists show reduced efficacy as compared to physiological agonists and can even act as antagonists in the presence of a full agonist. Here, we determined an X-ray crystal structure of the partial A2AAR agonist 2-amino-6-[(1H-imidazol-2-ylmethyl)sulfanyl]-4-p-hydroxyphenyl-3,5-pyridinedicarbonitrile (LUF5834) in complex with the A2AAR construct A2A-PSB2-bRIL, stabilized in its inactive conformation and being devoid of any mutations in the ligand binding pocket. The determined high-resolution structure (2.43 Å) resolved water networks and crucial binding pocket interactions. A direct hydrogen bond of the p-hydroxy group of LUF5834 with T883.36 was observed, an amino acid that was mutated to alanine in the most frequently used A2AAR crystallization constructs thus preventing the discovery of its interactions in most of the previous A2AAR co-crystal structures. G protein dissociation studies confirmed partial agonistic activity of LUF5834 as compared to that of the full agonist N-ethylcarboxamidoadenosine (NECA). In contrast to NECA, the partial agonist was still able to bind to the receptor construct locked in its inactive conformation by an S913.39K mutation, although with an affinity lower than that at the native receptor. This could explain the compound’s partial agonistic activity: while full A2AAR agonists bind exclusively to the active conformation, likely following conformational selection, partial agonists bind to active as well as inactive conformations, showing higher affinity for the active conformation. This might be a general mechanism of partial agonism also applicable to other GPCRs. Less
Crimean Congo hemorrhagic fever virus CCHFV is a tick-borne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we used structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn and ... More
Crimean–Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause severe disease in humans with case fatality rates of 10–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we used structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-EM structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
Rhomboid intramembrane serine proteases have been implicated in several pathologies and emerge as attractive pharmacological target candidates The most potent and selective rhomboid inhibitors available to date are peptidyl -ketoamides but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood This gap together with the lack of suitable in vitro models hinders ketoamide development for relevant eukaryotic rhomboid enzymes Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG human mitochondrial rhomboid PARL and human RHBDL ... More
Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipid cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesised here containing branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2. Less
Vitamin B cobalamin or Cbl functions as a cofactor in two important enzymatic processes in human cells and life is not sustainable without it B is obtained from food and travels from the stomach through the intestine and into the bloodstream by three B -transporting proteins salivary haptocorrin HC gastric intrinsic factor IF and transcobalamin TC which all bind B with high affinity and require proteolytic degradation to liberate Cbl After intracellular delivery of dietary B Cbl in the aquo hydroxo-Cbl HOCbl form can coordinate various nucleophiles e g glutathione GSH giving rise to glutathionylcobalamin GSCbl a naturally-occurring form of ... More
Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxo-Cbl (HOCbl) form can coordinate various nucleophiles, e.g., glutathione (GSH), giving rise to glutathionylcobalamin (GSCbl), a naturally-occurring form of vitamin B12. Currently there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12-transporter and GSCbl, which compared to HOCbl, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, HC and IF are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl. Less
Light-driven sodium pumps NaRs are unique ion-transporting microbial rhodopsins The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base We thoroughly characterize a member of this subgroup namely the protein ErNaR from Erythrobacter sp HL- and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity which is in contrast to previously studied NaRs ... More
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics. Less
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis Protein phosphatase PP A is an essential regulator of cell growth One scaffold subunit A binds to a catalytic subunit C to form a core AC heterodimer which together with one of many regulatory B subunits forms the active trimeric enzyme The combinatorial number of distinct PP A complexes is large which results in diverse substrate specificity and subcellular localization The detailed mechanism of PP A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP A C are conflicting A better ... More
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissect PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes. Less
Sphingolipid activator protein B saposin B SapB is an essential activator of globotriaosylceramide Gb catabolism by -galactosidase A However the manner by which SapB stimulates -galactosidase A activity remains unknown To uncover the molecular mechanism of SapB presenting Gb to -galactosidase A we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole Gb -NBD to a series of biochemical and structural assays involving SapB First we showed that SapB stably binds Gb -NBD using a fluorescence equilibrium binding assay isolates Gb -NBD from micelles and facilitates -galactosidase A cleavage of Gb -NBD in vitro Second we crystallized SapB in the presence of Gb -NBD ... More
Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and α-galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to α-galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology. Less
Evolution leads to conservation of amino acid residues in protein families Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure Surprisingly proline residues that are highly conserved in class A -lactamases were found to tolerate various substitutions without large losses in enzyme activity We investigated the roles of three conserved prolines at positions and in the -lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time For the variant Pro Thr the crystal ... More
Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme. Less
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria which are valuable for bioenergy development and biotechnology Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins scaffoldins and dockerin modules in enzymes Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes tandem dockerins also exist albeit very rare in some cellulosomal components whose assembly and functional roles are currently unclear In this study we characterized the structure and mode of assembly of a tandem bimodular double-dockerin which is connected to a putative S protease in ... More
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components. Less
Mycobacterium tuberculosis Mtb adapt to various host environments and utilize a variety of sugars and lipids as carbon sources Among these sugars maltose and trehalose also play crucial role in bacterial physiology and virulence However some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known Here we structurally and functionally characterized a conserved hypothetical gene Rv We determined the crystal structure of Rv at resolution The crystal structure revealed that Rv adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins Our comparative structural analysis suggested that Rv could perform either ... More
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β-phosphoglucomutase (β-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β-PGM activity and hence, Rv3400 encodes for β-PGM in Mtb. Our data also confirm that Mtb β-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β-PGM converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β-PGM in the physiology of Mtb. Less
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability selectivity metabolic stability and rapid clearance of peptides Here we describe the rational design synthesis and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead We have identified potent and selective inhibitors of TMPRSS matriptase hepsin and HGFA and demonstrated their improved protease selectivity metabolic stability and pharmacokinetic PK properties We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD b bound to matriptase revealing an unexpected binding conformation Cyclic biphenyl ether VD ... More
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery. Less
The CD IGFBP axis proteins are key factors expressed in endothelial cells EC that mediate EC angiogenesis and migration Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions However the interactions of these proteins with each other remain unclear In this study we determined a partial structure of the human CD IGFBP complex comprising the EGF domain of CD and the IB domain of IGFBP Mutagenesis studies confirmed interactions and specificities Cellular and mouse tumor studies demonstrated the physiological relevance of the CD IGFBP interaction in EC angiogenesis ... More
The CD93/IGFBP7 axis proteins are key factors expressed in endothelial cells (EC) that mediate EC angiogenesis and migration. Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions. However, the interactions of these proteins with each other remain unclear. In this study, we determined a partial structure of the human CD93–IGFBP7 complex comprising the EGF1 domain of CD93 and the IB domain of IGFBP7. Mutagenesis studies confirmed interactions and specificities. Cellular and mouse tumor studies demonstrated the physiological relevance of the CD93–IGFBP7 interaction in EC angiogenesis. Our study provides leads for the development of therapeutic agents to precisely disrupt unwanted CD93–IGFBP7 signaling in the tumor microenvironment. Additionally, analysis of the CD93 full-length architecture provides insights into how CD93 protrudes on the cell surface and forms a flexible platform for binding to IGFBP7 and other ligands. Less
Plant pathogens secrete proteins known as effectors that function in the apoplast or inside plant cells to promote virulence Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity Despite their importance our general understanding of fungal effector function and recognition by immunity receptors remains poor One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function In recent years several studies have demonstrated that fungal effectors can be grouped into structural classes despite significant sequence variation and existence across taxonomic ... More
Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein X-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant–fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens. Less
Periplasmic solute-binding proteins SBPs are key ligand recognition components of bacterial ATP-binding cassette ABC transporters that allow bacteria to import nutrients and metabolic precursors from the environment Periplasmic SBPs comprise a large and diverse family of proteins of which only a small number have been empirically characterized In this work we identify a set of unique uncharacterized proteins within the SBP bac family that are found in conserved operons comprising genes encoding i ABC transport systems and ii putative amidases from the FmdA AmdA family From these uncharacterized SBP bac proteins we characterize a representative periplasmic SBP from Mesorhizobium sp ... More
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization. Less
Infected cell protein ICP is an immediate-early regulatory protein of herpes simplex virus HSV- that possesses E ubiquitin ligase activity ICP transactivates viral genes in part through its C-terminal dimer domain residues Deletion of this dimer domain results in reduced viral gene expression lytic infection and reactivation from latency Since ICP s dimer domain is associated with its transactivation activity and efficient viral replication we wanted to determine the structure of this specific domain The C-terminus of ICP was purified from bacteria and analyzed by X-ray crystallography to solve its structure Each subunit or monomer in the ICP dimer is ... More
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555–767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0’s dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β-strands and two α-helices. Interestingly, two adjacent β-strands from one monomer “reach” into the adjacent subunit during dimer formation, generating two β-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β-strands of each dimer, creating a “stacking” of the β-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle. Less
Osteoclasts are over-activated as we age which results in bone loss Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts indicating that Src function is essential in osteoclasts G-protein-coupled receptors GPCRs are the targets for of approved drugs but it is still unclear how GPCRs regulate Src kinase activity Here we reveal that GPR activation by its natural ligand Kisspeptin- Kp- causes Dusp to dephosphorylate Src at Tyr Mechanistically Gpr recruits both active Src and the Dusp phosphatase at its proline arginine-rich motif in its C terminus We show that Kp- binding to Gpr ... More
Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation. Less
Melanocortin receptor MC -R antagonists are actively sought for treating cancer cachexia We determined the structures of complexes with PG- and SBL-MC- These peptides differ from SHU by substituting His with Pro and inserting Gly or Arg The structures revealed two subpockets at the TM -TM -TM domains separated by N Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study Most ligands retained the SHU potency but several SBL-MC- -derived peptides significantly enhanced MC -R selectivity over MC -R by - to -fold We also investigated MC -R coupling to the K ... More
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders. Less
The adenosine subfamily G protein-coupled receptors A AR and A BR have been identified as promising cancer immunotherapy candidates One of the A AR A BR dual antagonists AB has progressed to a phase II clinical trial to treat rectal cancer However the precise mechanism underlying its dual-antagonistic properties remains elusive Herein we report crystal structures of the A AR complexed with AB and a selective A AR antagonist The structures revealed a common binding mode on A AR wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets In contrast the cAMP assay and A ... More
The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2–118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2–118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy. Less
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels This process is mediated by the parasite s polymorphic erythrocyte membrane protein PfEMP adhesion proteins A subset of PfEMP variants that bind human endothelial protein C receptor EPCR through their CIDR domains is responsible for severe malaria pathogenesis A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP variants Here we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of CIDR domains representing five of ... More
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite’s polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Less
Monoolein-based liquid crystal phases are established media that are researched for various biological applications including drug delivery While water is the most common solvent for self-assembly some ionic liquids ILs can support lipidic self-assembly However currently there is limited knowledge of IL-lipid phase behavior in ILs In this study the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly namely ethylammonium nitrate ethanolammonium nitrate ethylammonium formate ethanolammonium formate ethylammonium acetate and ethanolammonium acetate These ILs were selected to identify specific ion effects on monoolein self-assembly specifically increasing the alkyl chain length ... More
Monoolein-based liquid crystal phases are established media that are researched for various biological applications, including drug delivery. While water is the most common solvent for self-assembly, some ionic liquids (ILs) can support lipidic self-assembly. However, currently, there is limited knowledge of IL-lipid phase behavior in ILs. In this study, the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly, namely ethylammonium nitrate, ethanolammonium nitrate, ethylammonium formate, ethanolammonium formate, ethylammonium acetate, and ethanolammonium acetate. These ILs were selected to identify specific ion effects on monoolein self-assembly, specifically increasing the alkyl chain length of the cation or anion, the presence of a hydroxyl group in the cation, and varying the anion. The lyotropic liquid crystal phases with 20–80 wt. % of monoolein were characterized over a temperature range from 25 to 65 °C using synchrotron small angle x-ray scattering and cross-polarized optical microscopy. These results were used to construct partial phase diagrams of monoolein in each of the six protic ILs, with inverse hexagonal, bicontinuous cubic, and lamellar phases observed. Protic ILs containing the ethylammonium cation led to monoolein forming lamellar and bicontinuous cubic phases, while those containing the ethanolammonium cation formed inverse hexagonal and bicontinuous cubic phases. Protic ILs containing formate and acetate anions favored bicontinuous cubic phases across a broader range of protic IL concentrations than those containing the nitrate anion. Less
TP is the most frequently mutated gene in human cancer While it is well understood that the ability of p to act as a transcription factor is required for tumor suppression the key target genes downstream of p required for tumor suppression are still incompletely understood We first set out to characterize a rare African-specific germline variant of TP in the DNA binding domain Tyr His Y H Although we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes Y H mice develop spontaneous cancers ... More
TP53 is the most frequently mutated gene in human cancer. While it is well understood that the ability of p53 to act as a transcription factor is required for tumor suppression, the key target genes downstream of p53 required for tumor suppression are still incompletely understood. We first set out to characterize a rare, African-specific, germline variant of TP53 in the DNA binding domain, Tyr107His (Y107H). Although we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes, Y107H mice develop spontaneous cancers and metastases. We identified the p53 target gene, PADI4, to be exquisitely sensitive to p53 mutation, and loss of PADI4 is seen in Y107H and other transcriptionally competent p53 hypomorphs. PADI4 is a regulator of histone modification and gene transcription via citrullination, which is the process of deiminating arginine to the non-natural amino acid citrulline. Our TCGA analysis reveals PADI4 is downregulated or mutated in multiple human cancers. Surprisingly, we show that PADI4 is sufficient to suppress tumor growth and sensitize wild-type p53 cells to chemotherapeutics. We further show that PADI4 is potently tumor suppressive in vivo, and complete tumor suppression by PADI4 requires an intact immune system. We find PADI4 enhances the transactivation of p53 targets and genes involved in immune activation. In addition, we identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. We have further found that PADI4 interacts and modifies p53 via citrullination at key residues within the oligomerization and C-terminal domain of p53. PADI4 colocalizes with p53 on chromatin at non-canonical p53 target genes and genes devoid of a p53 response element. Citrullination of p53 may alter p53 function through enhanced oligomerization or binding of p53 to DNA. The findings from this study reveal PADI4 as not only a key target gene of p53, but a core regulator of p53 activity and target specificity through a novel protein modification. This work highlights the need to reassess the role of PADI4 in cancer, and provides insight into critical downstream target genes important for tumor suppression by p53. Less
Membrane proteins are indispensable for every living organism yet their structural organization remains underexplored Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography which have significantly increased the structural coverage of membrane proteins across various kingdoms certain scientific methods such as time-resolved crystallography still mostly rely on crystallization techniques such as lipidic cubic phase LCP or in meso crystallization In this study we present an open-access blueprint for a humidity control chamber designed for LCP in meso crystallization experiments using a Gryphon crystallization robot Using this chamber we have obtained crystals of a transmembrane aspartate transporter ... More
Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community. Less
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching Despite favorable fluorescence properties use of StayGold as a fluorescent tag is limited because it forms a natural dimer Here we report the structure of StayGold and generate a derivative mStayGold that retains the brightness and photostability of the original protein while being fully monomeric
NADH cytochrome b oxidoreductase Ncb or is a cytosolic ferric reductase implicated in diabetes and neurological conditions Ncb or comprises cytochrome b b and cytochrome b reductase b R domains separated by a CHORD-Sgt CS linker domain Ncb or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme While full-length human Ncb or has proven resistant to crystallization we have succeeded in obtaining high-resolution atomic structures of the b domain and a construct containing the CS and b R domains CS b R Ncb or also contains an N-terminal intrinsically ... More
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. Less
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins yet have very similar spectra when in the oxidized state Recently we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue Q V or red I V A Q shifts of fluorescence emission maxima Here we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q V an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I V A Q variant Consequently in Q V excitation emission and phosphorescence spectra are shifted whereas in I V A ... More
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties. Less
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems However it is unclear how the antenna transfers energy efficiently to these photosystems We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein each composed of two protomers but with different quaternary structures arising from a diverse subunit family We report crystal structures of the major phycobiliprotein from each spectrotype Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors These are supplemented by a newly discovered open-braced form ... More
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αβ protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one β subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein. Less
Kinases are important therapeutic targets and their inhibitors are classified according to their mechanism of action which range from blocking ATP binding to covalent inhibition Here a mechanism of inhibition is highlighted by capturing p -activated kinase PAK in an intermediate state of activation using an Affimer reagent that binds in the P pocket PAK was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells Silencing of PAK resulted in reduced cell number G S arrest and enlargement of cells suggesting it to be important in urothelial cancer cell line survival and proliferation Affimer reagents were isolated ... More
Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible. Less
Poly ADP-ribose polymerase PARP inhibitors are used in the clinic to treat BRCA-deficient breast ovarian and prostate cancers As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH there is considerable interest in the development of highly specific small molecule DNPH inhibitors Here we present X-ray crystal structures of dimeric DNPH bound to its substrate hydroxymethyl deoxyuridine monophosphate hmdUMP Direct interaction with the hydroxymethyl group is important for substrate positioning while conserved residues surrounding the base facilitate target discrimination Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation ... More
Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme’s catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery. Less
Biochemical Properties of a Promising Milk-Clotting Enzyme, Moose (Alces alces) Recombinant Chymosin
Moose Alces alces recombinant chymosin with a milk-clotting activity of AU mL was synthesized in the Kluyveromyces lactis expression system After precipitation with ammonium sulfate and chromatographic purification a sample of genetically engineered moose chymosin with a specific milk-clotting activity of AU mg was obtained which was used for extensive biochemical characterization of the enzyme The threshold of the thermal stability of moose chymosin was C its complete inactivation occurred after heating at C The total proteolytic activity of moose chymosin was A units The ratio of milk-clotting and total proteolytic activities of the enzyme was The Km kcat and ... More
Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A280 units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The Km, kcat and kcat/Km values of moose chymosin were 4.7 μM, 98.7 s−1, and 21.1 μM−1 s−1, respectively. The pattern of change in the coagulation activity as a function of pH and Ca2+ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg2+, Zn2+, Co2+, Ba2+, Fe2+, Mn2+, Ca2+, and Cu2+ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers. Less
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides The metabolism of - -linked cello-oligosaccharides is initiated by three enzymes i e the cellodextrin phosphorylase Cdp the cellobiose phosphorylase Cbp and the -glucosidase A BglA in C thermocellum In comparison how the oligosaccharides containing other kinds of linkage are utilized is rarely understood In this study we found that BglA could hydrolyze the - -disaccharide laminaribiose with much higher activity than that against the - -disaccharide cellobiose The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking Genetic deletions of BglA and Cbp ... More
The thermophilic bacterium Clostridium thermocellum efficiently degrades polysaccharides into oligosaccharides. The metabolism of β-1,4-linked cello-oligosaccharides is initiated by three enzymes, i.e., the cellodextrin phosphorylase (Cdp), the cellobiose phosphorylase (Cbp), and the β-glucosidase A (BglA), in C. thermocellum. In comparison, how the oligosaccharides containing other kinds of linkage are utilized is rarely understood. In this study, we found that BglA could hydrolyze the β-1,3-disaccharide laminaribiose with much higher activity than that against the β-1,4-disaccharide cellobiose. The structural basis of the substrate specificity was analyzed by crystal structure determination and molecular docking. Genetic deletions of BglA and Cbp, respectively, and enzymatic analysis of cell extracts demonstrated that BglA is the key enzyme responsible for laminaribiose metabolism. Furthermore, the deletion of BglA can suppress the expression of Cbp and the deletion of Cbp can up-regulate the expression of BglA, indicating that BglA and Cbp have cross-regulation and BglA is also critical for cellobiose metabolism. These insights pave the way for both a fundamental understanding of metabolism and regulation in C. thermocellum and emphasize the importance of the degradation and utilization of polysaccharides containing β-1,3-linked glycosidic bonds in lignocellulose biorefinery. Less
Laboratory production of recombinant mammalian proteins particularly antibodies requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications Transient gene expression TGE in the suspension-adapted Chinese Hamster Ovary CHO cell lines has become the method of choice for this task The antibodies can be secreted into the media which facilitates subsequent purification and can be glycosylated However in general protein production in CHO cells is expensive and may provide variable outcomes namely in laboratories without previous experience While achievable yields may be influenced by the nucleotide sequence there are other aspects of the process which offer ... More
Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Less
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats However their widespread utilization has contributed to the emergence and spread of antibiotic resistance which now poses a significant public health challenge Streptomyces bacterium produce diverse secondary metabolites with antibacterial antifungal antiviral antitumoral and immunosuppressant activities Among these compounds is echinomycin a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps A gene called ecm was identified in the echinomycin biosynthetic gene cluster which provides echinomycin self-resistance Ecm recognizes DNA duplexes ... More
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes that contain echinomycin, and neutralizes its toxicity through a yet undetermined mechanism. To shed light on this process, we are determining the crystal structure of Ecm16 containing ATP. We introduced strategic mutations in the nucleotide binding site of Ecm16 to prevent ATP hydrolysis. We have successfully cloned, expressed, and purified the recombinant Ecm16 E399Q,E708Q double mutant protein. We have solved the structure of this protein using X-ray Crystallography at a resolution of 2.07 Å. However, our crystal structure contained ADP instead of the expected ATP. We propose and alternative experimental strategies for structure determination of ATP-containing Ecm16. Less
The Dictyostelium discoideum dye-decolorizing peroxidase DdDyP is a newly discovered peroxidase which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily DdDyP catalyzes the H O -dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass holding promise for potential industrial and biotechnological applications To study the molecular mechanism of DdDyP highly pure and functional protein with a natively incorporated heme is required however obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors Alternatively ... More
The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis. Less
TP is the most frequently mutated gene in cancer yet key target genes for p -mediated tumor suppression remain unidentified Here we characterize a rare African-specific germline variant of TP in the DNA-binding domain Tyr His Y H Nuclear magnetic resonance and crystal structures reveal that Y H is structurally similar to wild-type p Consistent with this we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes this includes the epigenetic modifier PADI which deiminates arginine to the nonnatural amino acid citrulline Surprisingly we show ... More
TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. Less
The small size and flexibility of G protein-coupled receptors GPCRs have long posed a significant challenge to determining their structures for research and therapeutic applications Single particle cryogenic electron microscopy cryoEM is often out of reach due to the small size of the receptor without a signaling partner Crystallization of GPCRs in lipidic cubic phase LCP often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers Here we determine the previously unknown structure of the human vasopressin B receptor V BR ... More
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods. Less
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp - HaDHR is a member of a small family of dehydrogenases containing a covalently attached FMN and the only member so far identified to date that does not exhibit substrate inhibition In this study we present the resolution crystal structure of HaDHR This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators Alanine was identified as the exit point of electrons from the Fe S cluster The enzyme was modified with a Ser Cys mutation to facilitate covalent attachment of a ferrocene moiety When modified ... More
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators. Less
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted compared to mesophilic orthologs In several cases the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation In the psychrophilic -amylase from an Antarctic bacterium the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature Here we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations ... More
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog. Less
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation T- domain responsible for the delivery of the catalytic domain into the cell Here we use a combination of computational constant-pH Molecular Dynamics simulations and experimental NMR circular dichroism and fluorescence spectroscopy along with the X-ray crystallography approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H Q H Q double mutant This replacement suppresses the acid-induced transition resulting in the retention of a more stable protein structure in solutions at ... More
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur. Less
We recently converted the GAF domain of NpR cyanobacteriochrome into near-infrared NIR fluorescent proteins FPs Unlike cyanobacterichrome which incorporates phycocyanobilin tetrapyrrole engineered NIR FPs bind biliverdin abundant in mammalian cells thus being the smallest scaffold for it Here we determined the crystal structure of the brightest blue-shifted protein of the series miRFP nano at resolution characterized its chromophore environment and explained the molecular basis of its spectral properties Using the determined structure we have rationally designed a red-shifted NIR FP termed miRFP nano with excitation at nm and emission at nm miRFP nano exhibits a small size of kDa enhanced ... More
We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities. Less
The high morbidity and mortality associated with SARS-CoV- infection the etiological agent of COVID- has had a major impact on global public health Significant progress has been made in the development of an array of vaccines and biologics however the emergence of SARS-CoV- variants and breakthrough infections are an ongoing major concern Furthermore there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV- and any emerging and re-emerging coronaviruses We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent ... More
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 μM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1e and 1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies. Less
The Gs protein-coupled adenosine A A receptor A AAR represents an emerging drug target for cancer immunotherapy The clinical candidate Etrumadenant was developed as an A AAR antagonist with ancillary blockade of the A BAR subtype It constitutes a unique chemotype featuring a poly-substituted -amino- -phenyl- -triazolylpyrimidine core structure Herein we report two crystal structures of the A AAR in complex with Etrumadenant obtained with differently thermostabilized A AAR constructs This led to the discovery of an unprecedented interaction a hydrogen bond of T with the cyano group of Etrumadenant T is mutated in most A AAR constructs used for ... More
The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations. Less
Cytotoxic-T-lymphocyte CTL mediated control of HIV- is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I HLA-I However the extent to which the presenting HLA allele contributes to this process is unknown Here we examine the CTL response to QW a highly networked epitope presented by the disease-protective HLA-B and disease-neutral HLA-B Despite robust targeting of QW in persons expressing either allele T cell receptor TCR cross-recognition of the naturally occurring variant QW S T is consistently reduced when presented by HLA-B but not by HLA-B Crystal structures show substantial conformational changes from QW -HLA to QW S T-HLA ... More
Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design. Less
In the DarTG toxin-antitoxin system the DarT toxin ADP-ribosylates single-stranded DNA ssDNA which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG DarG also binds DarT but the role of these interactions in DarT neutralization is unknown Here we report that the C-terminal domain of DarG DarG toxin-binding domain DarGTBD interacts with DarT to form a stoichiometric heterodimeric complex We determined the resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex The comparative structural analysis reveals that DarGTBD interacts with ... More
In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions. Less
Proteorhodopsins PRs bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins they play a significant role in life on the Earth A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name mirror proteorhodopsins from Sphingomonas paucimobilis SpaR shows cavity gate architecture of ... More
Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest. Less
SARS-CoV- and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including CL protease PL protease and RNA dependent RNA polymerase This dissertation summarizes the research findings related to CL protease inhibitors of SARS-CoV- and MERS-CoV A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor- CL protease complexes The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency Multiple inhibitors ... More
SARS-CoV-2 and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population. Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including 3CL protease, PL protease and RNA dependent RNA polymerase.
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
Influenza virus IV causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars Several influenza pandemics have occurred during the last century and estimated to have caused million deaths There are four genera of IV A IVA B IVB C IVC and D IVD with IVA being the most virulent to the human population Hemagglutinin HA is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell Here we have characterised the high-resolution structures of seven IVA HAs with one ... More
Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines. Less
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes It has two different enzymatic activities microbial cell walls destruction muramidase activity and dissolution of the stabilized fibrin isopeptidase activity Both activities are known to be inhibited by sodium chloride at near physiological concentrations but the structural basis remains unknown Here we present two crystal structures of destabilase including a -resolution structure in complex with sodium ion Our structures reveal the location of sodium ion between Glu Asp residues which were previously recognized as a glycosidase active site While sodium coordination with these amino acids may ... More
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development. Less
Transporters of the Nramp Natural resistance-associated macrophage protein family import divalent transition metal ions into cells of most organisms By supporting metal homeostasis Nramps prevent diseases and disorders related to metal insufficiency or overload Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site We present high-resolution structures of Deinococcus radiodurans Dra Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate Mn across conformations and by conserved networks of polar residues lining the inner and outer gates In addition a ... More
Transporters of the Nramp (Natural resistance-associated macrophage protein) family import divalent transition metal ions into cells of most organisms. By supporting metal homeostasis, Nramps prevent diseases and disorders related to metal insufficiency or overload. Previous studies revealed that Nramps take on a LeuT fold and identified the metal-binding site. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in three stable conformations of the transport cycle revealing that global conformational changes are supported by distinct coordination geometries of its physiological substrate, Mn2+, across conformations, and by conserved networks of polar residues lining the inner and outer gates. In addition, a high-resolution Cd2+-bound structure highlights differences in how Cd2+ and Mn2+ are coordinated by DraNramp. Complementary metal binding studies using isothermal titration calorimetry with a series of mutated DraNramp proteins indicate that the thermodynamic landscape for binding and transporting physiological metals like Mn2+ is different and more robust to perturbation than for transporting the toxic Cd2+ metal. Overall, the affinity measurements and high-resolution structural information on metal substrate binding provide a foundation for understanding the substrate selectivity of essential metal ion transporters like Nramps. Less
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine which is tightly bound in the protein inner cavity The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed many features of their functioning are yet unclear In particular which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role ... More
Hydromedusan photoproteins responsible for the bioluminescence of a variety of marine jellyfish and hydroids are a unique biochemical system recognized as a stable enzyme-substrate complex consisting of apoprotein and preoxygenated coelenterazine, which is tightly bound in the protein inner cavity. The binding of calcium ions to the photoprotein molecule is only required to initiate the light emission reaction. Although numerous experimental and theoretical studies on the bioluminescence of these photoproteins were performed, many features of their functioning are yet unclear. In particular, which ionic state of dioxetanone intermediate decomposes to yield a coelenteramide in an excited state and the role of the water molecule residing in a proximity to the N1 atom of 2-hydroperoxycoelenterazine in the bioluminescence reaction are still under discussion. With the aim to elucidate the function of this water molecule as well as to pinpoint the amino acid residues presumably involved in the protonation of the primarily formed dioxetanone anion, we constructed a set of single and double obelin and aequorin mutants with substitutions of His, Trp, Tyr, and Ser to residues with different properties of side chains and investigated their bioluminescence properties (specific activity, bioluminescence spectra, stopped-flow kinetics, and fluorescence spectra of Ca2+-discharged photoproteins). Moreover, we determined the spatial structure of the obelin mutant with a substitution of His64, the key residue of the presumable proton transfer, to Phe. On the ground of the bioluminescence properties of the obelin and aequorin mutants as well as the spatial structures of the obelin mutants with the replacements of His64 and Tyr138, the conclusion was made that, in fact, His residue of the Tyr-His-Trp triad and the water molecule perform the “catalytic function” by transferring the proton from solvent to the dioxetanone anion to generate its neutral ionic state in complex with water, as only the decomposition of this form of dioxetanone can provide the highest light output in the light-emitting reaction of the hydromedusan photoproteins. Less
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source thereby reducing the risk of kidney stone formation in host animals The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates Here we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations occluded and outward-facing states The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate The occluded pocket can accommodate ... More
An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis. Less
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes deaths annually in the United States prompting a need for vaccine development In addition to the important toxins TcdA and TcdB binary toxin CDT plays a significant role in the pathogenesis of certain C difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells However the mechanisms of CDT neutralization by antibodies have not been studied limiting our understanding of key epitopes for CDT antigen design Therefore we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically Here - and - resolution X-ray ... More
Clostridioides difficile is a Gram-positive opportunistic human pathogen that causes 15,000 deaths annually in the United States, prompting a need for vaccine development. In addition to the important toxins TcdA and TcdB, binary toxin (CDT) plays a significant role in the pathogenesis of certain C. difficile ribotypes by catalyzing the ADP-ribosylation of actin in host cells. However, the mechanisms of CDT neutralization by antibodies have not been studied, limiting our understanding of key epitopes for CDT antigen design. Therefore, we isolated neutralizing monoclonal antibodies against CDT and characterized their mechanisms of neutralization structurally and biochemically. Here, 2.5-Å and 2.6-Å resolution X-ray crystal structures of the antibodies BINTOXB/22 and BINTOXB/9, respectively, in complex with CDTb—the CDT subunit that forms a heptameric pore for the delivery of toxic CDTa enzyme into the host cytosol—showed that both antibodies sterically clash with adjacent protomers in the assembled heptamer. Assessment of trypsin-induced oligomerization of the purified CDTb protoxin in vitro showed that BINTOXB/22 and BINTOXB/9 prevented the assembly of di-heptamers upon prodomain cleavage. This work suggests that the CDT oligomerization process can be effectively targeted by antibodies, which will aid in the development of C. difficile vaccines and therapeutics. Less
Tyrosine kinases TKs play essential roles in signaling processes that regulate cell survival migration and proliferation Dysregulation of tyrosine kinases underlies many disorders including cancer cardiovascular and developmental diseases as well as pathologies of the immune system Ack and Brk are nonreceptor tyrosine kinases NRTKs best known for their roles in cancer Here we have biochemically characterized novel Ack and Brk mutations identified in patients with systemic lupus erythematosus SLE These mutations are the first SLE-linked polymorphisms found among NRTKs We show that two of the mutants are catalytically inactive while the other three have reduced activity To understand the ... More
Tyrosine kinases (TKs) play essential roles in signaling processes that regulate cell survival, migration, and proliferation. Dysregulation of tyrosine kinases underlies many disorders, including cancer, cardiovascular and developmental diseases, as well as pathologies of the immune system. Ack1 and Brk are nonreceptor tyrosine kinases (NRTKs) best known for their roles in cancer. Here, we have biochemically characterized novel Ack1 and Brk mutations identified in patients with systemic lupus erythematosus (SLE). These mutations are the first SLE-linked polymorphisms found among NRTKs. We show that two of the mutants are catalytically inactive, while the other three have reduced activity. To understand the structural changes associated with the loss-of-function phenotype, we solved the crystal structure of one of the Ack1 kinase mutants, K161Q. Furthermore, two of the mutated residues (Ack1 A156 and K161) critical for catalytic activity are highly conserved among other TKs, and their substitution in other members of the kinase family could have implications in cancer. In contrast to canonical gain-of-function mutations in TKs observed in many cancers, we report loss-of-function mutations in Ack1 and Brk, highlighting the complexity of TK involvement in human diseases. Less
We report the structural biochemical and functional characterization of the product of gene PA from Pseudomonas aeruginosa PAO The protein termed Pa Dps adopts the Dps subunit fold and oligomerizes into a nearly spherical -mer quaternary structure at pH or in the presence of divalent cations at neutral pH and above The -Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer coordinated by conserved His Glu and Asp residues In vitro the di-iron centers catalyze the oxidation of Fe utilizing H O not O as an oxidant suggesting Pa Dps functions to aid P aeruginosa ... More
We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps. Less
PT Formulatrix is one of the industries that produce automation tools or robots therefore companies are required to produce automation products that meet the needs of the world market PT Formulatrix has production divisions LH Liquid Handling and RI Rock Imager Examples of the production of automation equipment include NT Formulator for liquid handling and RI RI RI for rock imagers The difference lies in the shape and function of each product The problem focuses on the rock imager production process where production is made with a make to order system and the assembly process is parallel making the assembly ... More
PT. Formulatrix is one of the industries that produce automation tools or robots, therefore companies are required to produce automation products that meet the needs of the world market. PT. Formulatrix has 2 production divisions LH (Liquid Handling) and RI (Rock Imager). Examples of the production of automation equipment include, NT8, Formulator for liquid handling and RI 1000, RI 182, RI 54 for rock imagers. The difference lies in the shape and function of each product. The problem focuses on the rock imager production process, where production is made with a make to order system and the assembly process is parallel, making the assembly not smooth because you have to choose components, waiting for replacement components because they are damaged or missing to assemble. To improve the effective and efficient production process, replace the better production process. While production in liquid handling is said to be smooth because the production is small and the product is small compared to the rock imager. Assembling in parallel makes assemblers a hassle when assembling robots, because they have to sort out robot components in one container. Making assembly inefficient and ineffective. After collecting 5S data to support the kitting process in the rock imager assembling division, the process of assembling a one-table robot with components. As for the application of the 5S method applied at PT. Formulatrix Indonesia. The seiri method is applied to every division without exception, because in each division many mechanical equipment is found scattered on tables or on the floor. Seiton is a continuation of seiri, where the sorting results that have been carried out will be followed by the process of arranging the sorted equipment. Seiso at this stage, what is being done is the cleaning process. The cleaning that is done is cleaning the work area, such as the floor of the equipment used for the production process. At this stage it is more directed at the process of monitoring the 5S method that has been implemented. This stage is the last part of the 5S method. This section focuses more on how to get used to the application of this method. With the kitting process and the 5S method, it is hoped that assembling robots will be more efficient and effective, because waiting, sorting out parts, and looking for tools to assemble will be more optimal than the old process. With the kitting process, the efficiency value is better than the old process, from the previous 83% to 92%, which the researchers got from a trial at PT. Formulatrix Indonesia. Keywords: Rock Imager production process, 5S, Kitting. Less
Enzymatic pockets such as those of histone deacetylases HDACs are among the most favored targets for drug development However enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs which are often involved in distinct multisubunit complexes Here we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat and anti-psychedelic activity of an HDAC knockout in vivo We highlight that these phenotypes are achieved while modulating the expression of - and -fold fewer genes than enzymatic and genetic ... More
Enzymatic pockets such as those of histone deacetylases (HDACs) are among the most favored targets for drug development. However, enzymatic inhibitors often exhibit low selectivity and high toxicity due to targeting multiple enzyme paralogs, which are often involved in distinct multisubunit complexes. Here, we report the discovery and characterization of a non-enzymatic small molecule inhibitor of HDAC transcriptional repression functions with comparable anti-tumor activity to the enzymatic HDAC inhibitor Vorinostat, and anti-psychedelic activity of an HDAC2 knockout in vivo. We highlight that these phenotypes are achieved while modulating the expression of 20- and 80-fold fewer genes than enzymatic and genetic inhibition in the respective models. Thus, by achieving the same biological outcomes as established therapeutics while impacting a dramatically smaller number of genes, inhibitors of protein-protein interactions can offer important advantages in improving the selectivity of epigenetic modulators. Less
Opioid-related fatal overdoses have reached epidemic proportions Because existing treatments for opioid use disorders offer limited long-term protection accelerating the development of newer approaches is critical Monoclonal antibodies mAbs are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream reducing drug distribution across the blood-brain barrier thus preventing or reversing opioid toxicity We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone morphine fentanyl and nicotine To determine their binding mechanism we used X-ray crystallography to solve the structures of mAbs bound to their respective targets to resolution or higher Structural ... More
Opioid-related fatal overdoses have reached epidemic proportions. Because existing treatments for opioid use disorders offer limited long-term protection, accelerating the development of newer approaches is critical. Monoclonal antibodies (mAbs) are an emerging treatment strategy that targets and sequesters selected opioids in the bloodstream, reducing drug distribution across the blood-brain barrier, thus preventing or reversing opioid toxicity. We previously identified a series of murine mAbs with high affinity and selectivity for oxycodone, morphine, fentanyl, and nicotine. To determine their binding mechanism, we used X-ray crystallography to solve the structures of mAbs bound to their respective targets, to 2.2 Å resolution or higher. Structural analysis showed a critical convergent hydrogen bonding mode that is dependent on a glutamic acid residue in the mAbs’ heavy chain and a tertiary amine of the ligand. Characterizing drug-mAb complexes represents a significant step toward rational antibody engineering and future manufacturing activities to support clinical evaluation. Less
Chemical modifications of RNA have key roles in many biological processes N -methylguanosine m G is required for integrity and stability of a large subset of tRNAs The methyltransferase WD repeat-containing protein METTL WDR complex is the methyltransferase that modifies G in the variable loop of certain tRNAs and its dysregulation drives tumorigenesis in numerous cancer types Mutations in WDR cause human developmental phenotypes including microcephaly How METTL WDR modifies tRNA substrates and is regulated remains elusive Here we show through structural biochemical and cellular studies of human METTL WDR that WDR serves as a scaffold for METTL and the ... More
Chemical modifications of RNA have key roles in many biological processes1,2,3. N7-methylguanosine (m7G) is required for integrity and stability of a large subset of tRNAs4,5,6,7. The methyltransferase 1–WD repeat-containing protein 4 (METTL1–WDR4) complex is the methyltransferase that modifies G46 in the variable loop of certain tRNAs, and its dysregulation drives tumorigenesis in numerous cancer types8,9,10,11,12,13,14. Mutations in WDR4 cause human developmental phenotypes including microcephaly15,16,17. How METTL1–WDR4 modifies tRNA substrates and is regulated remains elusive18. Here we show, through structural, biochemical and cellular studies of human METTL1–WDR4, that WDR4 serves as a scaffold for METTL1 and the tRNA T-arm. Upon tRNA binding, the αC region of METTL1 transforms into a helix, which together with the α6 helix secures both ends of the tRNA variable loop. Unexpectedly, we find that the predicted disordered N-terminal region of METTL1 is part of the catalytic pocket and essential for methyltransferase activity. Furthermore, we reveal that S27 phosphorylation in the METTL1 N-terminal region inhibits methyltransferase activity by locally disrupting the catalytic centre. Our results provide a molecular understanding of tRNA substrate recognition and phosphorylation-mediated regulation of METTL1–WDR4, and reveal the presumed disordered N-terminal region of METTL1 as a nexus of methyltransferase activity. Less
Linear IgE epitopes play essential roles in persistent allergies including peanut and tree nut allergies Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success However the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach To overcome this problem we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein The vector was used to make overlapping peptides derived from peanut allergens Ara h ... More
Linear IgE epitopes play essential roles in persistent allergies, including peanut and tree nut allergies. Using chemically synthesized peptides attached to membranes and microarray experiments is one approach for determining predominant epitopes that has seen success. However, the overall expense of this approach and the inherent challenges in scaling up the production and purification of synthetic peptides precludes the general application of this approach. To overcome this problem, we have constructed a plasmid vector for expressing peptides sandwiched between an N-terminal His-tag and a trimeric protein. The vector was used to make overlapping peptides derived from peanut allergens Ara h 2. All the peptides were successfully expressed and purified. The resulting peptides were applied to identify IgE binding epitopes of Ara h 2 using four sera samples from individuals with known peanut allergies. New and previously defined dominant IgE binding epitopes of Ara h 2 were identified. This system may be readily applied to produce agents for component- and epitope-resolved food allergy diagnosis. Less
The advent of SARS-CoV- the causative agent of COVID- and its worldwide impact on global health have provided the impetus for the development of effective countermeasures that can be deployed against the virus including vaccines monoclonal antibodies and direct-acting antivirals DAAs Despite these efforts the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is a validated target for the discovery of SARS-CoV- therapeutics In this ... More
The advent of SARS-CoV-2, the causative agent of COVID-19, and its worldwide impact on global health, have provided the impetus for the development of effective countermeasures that can be deployed against the virus, including vaccines, monoclonal antibodies, and direct-acting antivirals (DAAs). Despite these efforts, the current paucity of DAAs has created an urgent need for the creation of an enhanced and diversified portfolio of broadly acting agents with different mechanisms of action that can effectively abrogate viral infection. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is a validated target for the discovery of SARS-CoV-2 therapeutics. In this report, we describe the structure-guided utilization of the cyclopropane moiety in the design of highly potent inhibitors of SARS-CoV-2 3CLpro, SARS-CoV-1 3CLpro, and MERS-CoV 3CLpro. High-resolution cocrystal structures were used to identify the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and unravel the mechanism of action. Aldehydes 5c and 11c inhibited SARS-CoV-2 replication with EC50 values of 12 and 11 nM, respectively. Furthermore, the corresponding aldehyde bisulfite adducts 5d and 11d were equipotent with EC50 values of 13 and 12 nM, respectively. The safety index (SI) values for compounds 5c/11c and 5d/11d ranged between 7692 and 9090. Importantly, aldehydes 5c/11c and bisulfite adducts 5d/11d potently inhibited MERS-CoV 3CLpro with IC50 values of 80 and 120 nM, and 70 and 70 nM, respectively. Likewise, compounds 5c/11c and 5d/11d inhibited SARS-CoV-1 with IC50 values of 960 and 350 nM and 790 and 240 nM, respectively. Taken together, these studies suggest that the inhibitors described herein have low cytotoxicity and high potency and are promising candidates for further development as broad-spectrum direct-acting antivirals against highly pathogenic coronaviruses. Less
Crystallization of proteins is a critical step in structural biology biopharmaceutical industry and materials science Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput reduced consumption of reagents and lower cost However current mirofluidic approches generally lack the module for high-speed data analysis ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano- nano-liter-scaled volume To address the issues we propose a deep learning-aided programmable microliter-droplet system which allows the high-throughput screening of time-resolved protein crystallization ... More
Crystallization of proteins is a critical step in structural biology, biopharmaceutical industry and materials science. Microfluidic technology has emerged as a promising tool for screening the crystallization conditions with advantages of increased throughput, reduced consumption of reagents and lower cost. However, current mirofluidic approches generally lack the module for high-speed data analysis, ignore the time-resolved changes of protein crystalline states or morphologies in the crystallization process and suffer from inconsistency after scaling up due to the subnano-/nano-liter-scaled volume. To address the issues, we propose a deep learning-aided programmable microliter-droplet system, which allows the high-throughput screening of time-resolved protein crystallization in microliter scale. Based on the system, a series of temporal phase diagrams are acquired, which reveal the time-resolved crystallization of target proteins under different crystallization conditions. They provide precise guidance on the scale-up experiment (∼93 % in consistency), and help gain insight into the kinetic characteristics of protein crystallization. Less
Mitogen-activated protein kinase MAPK phosphatase MKP is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure In previous work we identified Compound -dimethyl- - - methylthio - -dihydrothieno -h quinazolin- -yl thio butan- -one as the lead compound of a novel class of MKP inhibitors In this work we explore the structure-activity relationship for inhibition of MKP through modifications to the scaffold and functional groups present in A series of ... More
Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease. Less
Applying rational design we developed kDa cyanobacteriochrome-based near-infrared NIR-I fluorescent protein miRFP nano miRFP nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues miRFP nano has maximal emission at nm and an emission tail in the short-wave infrared SWIR region allowing deep-penetrating off-peak fluorescence imaging in vivo The miRFP nano structure reveals the molecular basis of its red shift We demonstrate superiority of miRFP nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation
Coelenterazine-v CTZ-v a synthetic vinylene-bridged -extended derivative is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths However Ca -regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence Here we report the crystal structure of semi-synthetic Ca -discharged obelin-v bound with the reaction product determined at resolution Comparison of the crystal structure of Ca -discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure However the drastic changes in CTZ-binding ... More
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v. Less
The past fifty years have been marked by the surge of neurodegenerative diseases Unfortunately current treatments are only symptomatic Hence the search for new and innovative therapeutic targets for curative treatments becomes a major challenge Among these targets the adenosine A A receptor A AAR has been the subject of much research in recent years In this paper we report the design synthesis and pharmacological analysis of quinazoline derivatives as A AAR antagonists with high ligand efficiency This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM- More precisely we ... More
The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes. Less
Transmembrane ion transport is a key process in living cells Active transport of ions is carried out by various ion transporters including microbial rhodopsins MRs MRs perform diverse functions such as active and passive ion transport photo-sensing and others In particular MRs can pump various monovalent ions like Na K Cl I NO The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp PCC and is named Synechocystis halorhodopsin SyHR The structural study of SyHR may help to understand what makes an MR pump divalent ions Here we present the crystal structure ... More
Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics. Less
The transcription factor TEAD together with its coactivator YAP TAZ is a key transcriptional modulator of the Hippo pathway Activation of TEAD transcription by YAP has been implicated in a number of malignancies and this complex represents a promising target for drug discovery However both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules mainly due to a lack of druggable pockets TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition Here we employed ... More
The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03–69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03–69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03–69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03–69 led to an in vivo compatible compound MYF-03–176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration. Less
Colibactin a DNA cross-linking agent produced by gut bacteria is implicated in colorectal cancer Its biosynthesis uses a prodrug resistance mechanism a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm This activation is mediated by ClbP an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain Although the transmembrane domain is required for colibactin activation its role in catalysis is unclear Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP ... More
Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads. Less
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer CRC development Understanding colibactin s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota Here we report small molecule boronic acid inhibitors of colibactin biosynthesis Designed to mimic the biosynthetic precursor precolibactin these compounds potently inhibit the colibactin-activating peptidase ClbP Using biochemical assays and crystallography we show that they engage the ClbP binding pocket forming a covalent bond with the catalytic serine These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block ... More
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. Less
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane s and manipulate their infected host To date secretion systems have been identified including the type IX secretion system T SS associated with human avian and farmed-fish diseases As a bacterial secretion system the T SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria PorX is a highly conserved protein that regulates the transcription of essential T SS components and additionally mediates the function of T SS via direct interaction with PorL the rotary motor protein ... More
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported. Less
The multifunctional human Parkinson s disease protein PARK DJ is an attractive therapeutic target due to its link with early-onset Parkinson s disease upregulation in various cancers and contribution to chemoresistance However only a few compounds have been identified to bind PARK due to the lack of a dedicated chemical toolbox We report the creation of such a toolbox and showcase the application of each of its components The selective PARK submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys Installment of different dyes onto the inhibitor delivered two PARK probes The Rhodamine probe provides a ... More
The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors. Less
The main focus of this project was chicken protein MICAL which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton The prominent role of the MICAL protein is primarily associated with axon guidance as it destabilizes actin filaments through its oxidative activity We focused on elucidating the molecular mechanisms of chicken MICAL autoinhibition using molecular and structural biology methods together with new protein structure prediction methods Chicken MICAL was produced in Sf insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL protein ... More
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis. Less
Modulators of the G protein-coupled A A adenosine receptor A AAR have been considered promising agents to treat Parkinson s disease inflammation cancer and central nervous system disorders Herein we demonstrate that a thiophene modification at the C position in the common adenine scaffold converted an A AAR agonist into an antagonist We synthesized and characterized a novel A AAR antagonist LJ- with Ki nM X-ray crystallographic structures of in complex with two thermostabilized A AAR constructs were solved at and resolutions In contrast to A AAR agonists which simultaneously interact with both Ser and His only transiently contacts His ... More
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson’s disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds. Less
Akt is a Ser Thr protein kinase that plays a central role in metabolism and cancer Regulation of Akt s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology PH domain and its kinase domain that can be relieved by C-tail phosphorylation PH domain mutant E K Akt is a well-established oncogene Previously we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds Chu et al Here we discover unexpectedly that a single mutation R A Akt exhibits intensified ... More
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt’s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver. Less
The bioactive lysophospholipid sphingosine- -phosphate S P acts via five different subtypes of S P receptors S PRs - S P - S P is predominantly expressed in nervous and immune systems regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders as well as carcinogenesis Several S PR therapeutic drugs have been developed to treat these diseases however they lack receptor subtype selectivity which leads to side effects In this article we describe a resolution room temperature crystal structure of the human S P receptor in complex with a selective ... More
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family. Less
N-Methylated amino acids N-MeAAs are privileged residues of naturally occurring peptides critical to bioactivity However de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA By reconfiguring the tRNA s T-stem region to compensate and tune the EF-Tu affinity we conducted Random nonstandard Peptides Integrated Discovery RaPID display of a macrocyclic peptide MCP library containing six different N-MeAAs We have here devised a pool-and-split enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate ... More
N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA’s T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a “pool-and-split” enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cisN-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library. Less
Microbial rhodopsins are light-sensitive transmembrane proteins evolutionary adapted by various organisms like archaea bacteria simple eukaryote and viruses to utilize solar energy for their survival A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures which can be primarily obtained by X-ray crystallography This technique however requires high-quality crystals growing of which is a great challenge especially in case of membrane proteins In this chapter we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases also known as in meso approach In particular we ... More
Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins. In this chapter, we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases, also known as in meso approach. In particular, we describe in detail the methods of crystallization using lipidic cubic phase to grow both large crystals optimized for traditional crystallographic data collection and microcrystals for serial crystallography. Less
The technique of cryogenic-electron microscopy cryo-EM has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species This report describes the structural characterization of a fully active human apelin receptor APJR complexed with heterotrimeric G protein observed in both and stoichiometric ratios We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation Protein preparations in the presence of the endogenous peptide ligand ELA or a synthetic small molecule both demonstrate these mixed stoichiometric states Structural differences in G protein engagement between dimeric and monomeric ... More
The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling. Less
The novel coronavirus pandemic whose first outbreak was reported in December in Wuhan China COVID- is caused by the severe acute respiratory syndrome coronavirus SARS-CoV- Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells which can elicit acute respiratory distress syndrome ARDS characterized by the rapid onset of widespread inflammation the so-called cytokine storm In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells DCs are seen as a response to the viral infection DCs are critical players in the development of the acute lung ... More
The novel coronavirus pandemic, whose first outbreak was reported in December 2019 in Wuhan, China (COVID-19), is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Tissue damage caused by the virus leads to a strong immune response and activation of antigen-presenting cells, which can elicit acute respiratory distress syndrome (ARDS) characterized by the rapid onset of widespread inflammation, the so-called cytokine storm. In many viral infections the recruitment of monocytes into the lung and their differentiation to dendritic cells (DCs) are seen as a response to the viral infection. DCs are critical players in the development of the acute lung inflammation that causes ARDS. Here we focus on the interaction of the ORF8 protein, a specific SARS-CoV-2 open reading frame protein, with dendritic cells (DCs). We show that ORF8 binds to dendritic cells, causes a pre-maturation of differentiating DCs, and induces the secretion of multiple pro-inflammatory cytokines by these cells. In addition, we identified dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) as a possible interaction partner of ORF8 on dendritic cells. Blockade of ORF8 signaling leads to reduced production of IL-1β, IL-6, IL-12p70, TNF-α, MCP-1 (CCL2), and IL-10 by dendritic cells. Analysis of patient sera with high anti-ORF8 antibody titers showed that there was nearly no neutralization of the ORF8 protein and its function. Therefore, a neutralizing antibody that has the capacity of blocking the cytokine and chemokine response mediated by ORF8 protein might be an essential and novel additional step in the therapy of severe SARS-CoV-2 cases. Less
Chitin is a biopolymer of N-acetyl-d-glucosamine with - -bond and is the main component of arthropod exoskeletons and the cell walls of many fungi Chitinase EC is an enzyme that hydrolyzes the - -bond in chitin and degrades chitin into oligomers It has been found in a wide range of organisms Chitinase from Gazyumaru Ficus microcarpa latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields However the enzyme s thermostability is an important factor chitinase is not thermostable enough to maintain its activity under the ... More
Chitin is a biopolymer of N-acetyl-d-glucosamine with β-1,4-bond and is the main component of arthropod exoskeletons and the cell walls of many fungi. Chitinase (EC 3.2.1.14) is an enzyme that hydrolyzes the β-1,4-bond in chitin and degrades chitin into oligomers. It has been found in a wide range of organisms. Chitinase from Gazyumaru (Ficus microcarpa) latex exhibits antifungal activity by degrading chitin in the cell wall of fungi and is expected to be used in medical and agricultural fields. However, the enzyme’s thermostability is an important factor; chitinase is not thermostable enough to maintain its activity under the actual application conditions. In addition to the fact that thermostable chitinases exhibiting antifungal activity can be used under various conditions, they have some advantages for the production process and long-term preservation, which are highly demanded in industrial use. We solved the crystal structure of chitinase to explore the target sites to improve its thermostability. We rationally introduced proline residues, a disulfide bond, and salt bridges in the chitinase using protein-engineering methods based on the crystal structure and sequence alignment among other chitinases. As a result, we successfully constructed the thermostable mutant chitinases rationally with high antifungal and specific activities. The results provide a useful strategy to enhance the thermostability of this enzyme family. Less
Antimicrobial resistance primarily caused by the overuse of antimicrobials such as antibiotics is becoming an increasing concern to public health To that end the global spread of the -lactamase OXA- is worrisome as it readily catalyzes the hydrolysis of -lactam drugs such as penicillins as well as our last resort carbapenems On the contrary OXA- exhibits only limited catalytic activity against rd generations cephalosporins like ceftazidime However naturally evolving variants and results from laboratory studies have shown that OXA- can expand its substrate profile conferring increased ceftazidime resistance Expansion of the substrate profile towards ceftazidime is seen to be accompanied ... More
Antimicrobial resistance, primarily caused by the overuse of antimicrobials such as antibiotics, is becoming an increasing concern to public health. To that end, the global spread of the -lactamase OXA-48 is worrisome, as it readily catalyzes the hydrolysis of -lactam drugs, such as penicillins as well as our “last resort” carbapenems. On the contrary, OXA-48 exhibits only limited catalytic activity against 3rd generations cephalosporins like ceftazidime. However, naturally evolving variants and results from laboratory studies have shown that OXA-48 can expand its substrate profile, conferring increased ceftazidime resistance. Expansion of the substrate profile towards ceftazidime is seen to be accompanied by a trade-off towards carbapenems and penicillins, greatly reducing OXA-48 ability to catalyze the hydrolysis of penicillins and carbapenems. Here, X-ray crystallography, steady-state enzyme kinetics and differential scanning fluorimetry were used to characterize and analyze wild type (wt) OXA-48:wt and two variants, OXA-48:F72L and OXA-48:A33V/K51E/F72L/S212A/T213A (OXA-48:Q5), where the latter two were evolved towards increased ceftazidime resistance. Steady-state enzyme kinetics revealed that the two mutants had increased catalytic ability to hydrolyze ceftazidime. Such increases in kcat/Km hypothesized to arise from increased flexibility of the -loop, which was observed in the OXA-48:Q5 X-ray crystal structure in complex with piperacillin, is in line with previous studies. Further supporting the hypothesis, urea dependent kinetics and thermostability measurements show that these mutants likely exhibit increased dynamical behavior that would aid ceftazidime binding. OXA-48:F72L showed a bigger urea dependence on the enzyme activity with no activity at 4 M urea, whereas OXA-48:wt and OXA-48:Q5 needed 6 M urea to become inactive. This suggested that OXA-48:F72L is more flexible, and that OXA-48:Q5 regains some resistance to chemical denaturing by urea. The pH dependency showed higher piperacillin activity at pH 7.2 compared to 5.2 and 9.2 for all three variants. The increase in ceftazidime activity came along with a functional trade-off against the penicillin piperacillin as well as reduced thermostability of (OXA-48:F72L: -6.5C/OXA-48:Q5: -6.4C) compared to wt OXA-48, which may be caused by sub-optimal substrate positioning within the active site of OXA-48:Q5. This work provides experimental evidence, that during evolution of OXA-48 towards increased ceftazidime activity, structural changes can arise, likely affecting the chemical environment within the active site, causing increased enzyme flexibility, and ultimately shaping functional trade-offs. Less
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical biophysical and bioinformatical approaches MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes as they facilitate the reorganization of the actin cytoskeleton MICAL- has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments However recent findings indicate that MICAL- can also serve as a signaling molecule using localized hydrogen peroxide production to regulate other downstream effectors Despite the consensus that MICAL- activity must ... More
This work aims to determine a model of the autoinhibition mechanism of MICAL proteins using biochemical, biophysical, and bioinformatical approaches. MICAL proteins are a group of flavin monooxygenases that play a key role in various cellular processes, as they facilitate the reorganization of the actin cytoskeleton. MICAL-1 has long been known for its vital role in axon guidance as an effector of repulsive signaling through oxidative destabilization of actin filaments. However, recent findings indicate that MICAL-1 can also serve as a signaling molecule, using localized hydrogen peroxide production to regulate other downstream effectors. Despite the consensus that MICAL-1 activity must be strictly regulated, the exact molecular mechanism of this regulation has not yet been described. In this work, we provide a novel model of MICAL-1 autoinibiton mechanism based on a comparison of steady-state kinetic experiments and molecular dynamics simulations between full-length MICAL-1 from Coturnix japonica and its truncated form lacking the C-terminal domain. In our model, we conclude that changes in MICAL-1 activity are the result of intramolecular protein interactions between the C-terminal and the monooxygenase domain. Furthermore, we rule out the role of MICAL-1 oligomerization in its activity regulation. Our work provides the basis for further research that will need to focus on a more detailed investigation of intramolecular interactions between the MICAL-1 domains. Less
The worldwide impact of the ongoing COVID- pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and or host targets SARS-CoV- C-like protease CLpro has emerged as a validated target for the discovery of SARS-CoV- therapeutics because of the pivotal role it plays in viral replication We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV- CLpro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space Inhibitors of both SARS-CoV- CLpro and MERS-CoV CLpro that exhibit nM potency and high ... More
The worldwide impact of the ongoing COVID-19 pandemic on public health has made imperative the discovery and development of direct-acting antivirals aimed at targeting viral and/or host targets. SARS-CoV-2 3C-like protease (3CLpro) has emerged as a validated target for the discovery of SARS-CoV-2 therapeutics because of the pivotal role it plays in viral replication. We describe herein the structure-guided design of highly potent inhibitors of SARS-CoV-2 3CLpro that incorporate in their structure novel spirocyclic design elements aimed at optimizing potency by accessing new chemical space. Inhibitors of both SARS-CoV-2 3CLpro and MERS-CoV 3CLpro that exhibit nM potency and high safety indices have been identified. The mechanism of action of the inhibitors and the structural determinants associated with binding were established using high-resolution cocrystal structures. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
Broadly HIV- neutralizing VRC -class antibodies bind the CD -binding site of Env and contain VH - derived heavy chains paired with light chains expressing five amino acid long CDRL s Their unmutated germline forms do not recognize HIV- Env and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding na ve B cells by the vaccine immunogens To address this point we examined Env-specific B cell receptor sequences from participants in the HVTN clinical trial Of all the sequences analyzed only one displayed homology to VRC -class antibodies but ... More
Broadly HIV-1–neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02–derived heavy chains paired with light chains expressing five–amino acid–long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line–targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans. Less
Chemically induced dimerization CID is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes The induced heterodimerization of FKBP fusion proteins by rapamycin and FK has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes Here we report the crystal structure of the AtFKBP FKBD in complex with rapamycin The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD The triene arm of rapamycin appears to ... More
Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes. Less
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins MPs at a molecular level High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs two different proton pumps and a sodium light-driven ion pump The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs a surface usually accommodating hydrophobic chains of annular lipids which are known structural and functional determinants for MPs In conformity ... More
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs – a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia. Less
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past years The most current and widespread of these is SARS-CoV- The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection We previously identified a neutralizing monoclonal antibody CV - that binds to the SARS-CoV- spike neutralizes the SARS-CoV- Beta variant comparably to the ancestral Wuhan Hu- ... More
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Less
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa dubbed the green rush In transgenic synthetic biology applications NphB is a promiscuous prenyltransferase from Streptomyces sp often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid CBGA the key precursor to many other cannabinoids However its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA Herein we demonstrated a detailed and extensive computational ... More
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years. This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa, dubbed the green rush. In transgenic synthetic biology applications, NphB is a promiscuous prenyltransferase from Streptomyces sp. often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid (CBGA), the key precursor to many other cannabinoids. However, its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA. Herein, we demonstrated a detailed and extensive computational structure-guided approach in identifying target residues of mutation for engineering NphB for optimal CBGA production. Our comprehensive computational workflow has led to the discovery of several highly regiospecific variants that produce CBGA exclusively, with the best-performing V49W/Y288P variant having a 13.6-fold yield improvement, outperforming all previous work on NphB enzyme engineering. We subsequently investigated the effects of these mutations by X-ray crystallographic studies of the mutant variants and performed molecular dynamics simulations to uncover an interplay of a H-bonding network and an optimal ligand orientation that favors the CBGA production over the side product. Collectively, this study not only recapitulates the utility of computational tools in informing and accelerating experimental design but also contributes to a better understanding of molecular mechanisms that govern enzyme regioselectivity and readily aids in cannabinoid synthetic biology production for future research into maximizing their therapeutic potential. Less
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase LCP To date there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability Here we report small-angle X-ray scattering measurements of a monoolein water mixture during continuous delivery using a high viscosity injector We observe both an alignment and modification of ... More
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase (LCP). To date, there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase. This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability. Here we report small-angle X-ray scattering measurements of a monoolein:water mixture during continuous delivery using a high viscosity injector. We observe both an alignment and modification of the LCP as a direct result of the injection process. The orientation of the cubic lattice with respect to the beam was estimated based on the anisotropy of the diffraction pattern and does not correspond to a single low order zone axis. The solvent fraction was also observed to impact the stability of the cubic phase during injection. In addition, depending on the distance traveled by the lipid after exiting the needle, the phase is observed to transition from a pure diamond phase () to a mixture containing both gyriod () and lamellar () phases. Finite element modelling of the observed phase behaviour during injection indicates that the pressure exerted on the lipid stream during extrusion accounts for the variations in the phase composition of the monoolein:water mixture. Less
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange altering the on off switch that forms the basis for their signaling functions Using X-ray crystallography nuclear magnetic resonance spectroscopy binding assays and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site ... More
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts. Less
L is a potent human monoclonal antibody mAb that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein PfCSP on malaria-infective sporozoites Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development Here we isolate mAbs clonally related to L and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity Pairing the L kappa light chain L with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs cross-react with NANP and more potently neutralize sporozoites in vivo ... More
L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs. Less
Macrophage migration inhibitory factor MIF is an inflammatory protein with various non-overlapping functions It is not only conserved in mammals but it is found in parasites fish and plants Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro as a catalytic base activates the receptors CD CXCR and CXCR has functional interactions in the cytosol and is reported to be a nuclease There is a solvent channel down its -fold axis with a recently identified gating residue as an allosteric site important for regulating to different extents the enzymatic activity and CD binding and signaling ... More
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight. Less
Pathogen effectors are crucial players during plant colonisation and infection Plant resistance mostly relies on effector recognition to activate defence responses Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment Here we examined the role of genetic diversity of the stem rust Puccinia graminis f sp tritici Pgt AvrSr gene in determining recognition by the corresponding wheat Sr resistance gene We solved the crystal structure of a natural variant of AvrSr and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence We report that AvrSr can ... More
Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment. Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence. We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties. This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations. Less
Base excision repair BER is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species BER is initiated by DNA glycosylases each of which repairs a particular class of base damage NTHL a bifunctional DNA glycosylase possesses both glycolytic and -lytic activities with a preference for oxidized pyrimidine substrates Defects in human NTHL drive a class of polyposis colorectal cancer We report the first X-ray crystal structure of hNTHL revealing an open conformation not previously observed in the bacterial orthologs In this conformation the six-helical barrel domain comprising the helix-hairpin-helix HhH DNA binding motif ... More
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1. Less
The COVID- pandemic is having a major impact on public health worldwide and there is an urgent need for the creation of an armamentarium of effective therapeutics including vaccines biologics and small-molecule therapeutics to combat SARS-CoV- and emerging variants Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is an attractive target for therapeutic intervention and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV- -specific antivirals We describe herein the results ... More
The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro. Less
Immunomodulatory drugs IMiDs thalidomide lenalidomide and pomalidomide Pom bind to cereblon CRBN and trigger proteasomal degradation of neo-substrates IKZF leading to multiple myeloma MM cell apoptosis Pomalidomide Pom also binds albeit weakly to p -related protein kinase PRPK aka TP RK an understudied kinase reported to be associated with poor prognosis in MM patients Here we developed a series of IMiDs based on Pom and conducted a structure-activity relationship SAR study to identify a potent and selective PRPK binder Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN and suggested specific derivatization to improve affinity ... More
Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies. Less
The worldwide outbreak of coronavirus disease COVID- caused by severe acute respiratory syndrome coronavirus SARS-CoV- has become an established global pandemic Alongside vaccines antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID- Here we report the discovery and characterization of PF- an orally bioavailable SARS-CoV- main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles PF- has demonstrated oral activity in a mouse- adapted SARS-CoV- model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase ... More
The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse- adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. Less
Current coronavirus CoV vaccines primarily target immunodominant epitopes in the S subunit which are poorly conserved and susceptible to escape mutations thus threatening vaccine efficacy Here we use structure-guided protein engineering to remove the S subunit from the Middle East respiratory syndrome MERS -CoV spike S glycoprotein and develop stabilized stem SS antigens Vaccination with MERS SS elicits cross-reactive -CoV antibody responses and protects mice against lethal MERS-CoV challenge High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies Among them antibody IgG binds with high affinity to both MERS-CoV ... More
Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies. Less
CD is a tumor necrosis factor TNF receptor which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD Activation of the CD receptor provides a costimulatory signal to promote T cell B cell and NK cell activity to facilitate antitumor and anti-infection immunity Aberrant increased and focused expression of CD on many tumor cells renders CD an attractive therapeutic target for direct tumor killing However despite their use as drug targets to treat cancers the molecular basis and atomic details of CD and CD interaction remain elusive Here we report the crystal structure of human CD in ... More
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction. Less
Medium-chain triglycerides MCTs are an emerging choice to treat neurodegenerative disorders such as Alzheimer s disease They are triesters of glycerol and three medium-chain fatty acids such as capric C and caprylic C acids The availability of C C methyl esters C C ME from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs However there are few reports on enzymes that can efficiently hydrolyse C C ME to industrial specifications Here we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus LTL which hydrolyses C ... More
Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer’s disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8–C10 methyl esters (C8–C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8–C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8–C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8–C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1’s well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs. Less
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV- envelope glycoprotein Env is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies NAbs Here we analyzed monoclonal antibodies mAbs isolated from non-human primates NHPs immunized with variants of a native flexibly linked NFL HIV- Env stabilized trimer derived from the tier clade C strain The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from to g ml IC Structural characterization using negative-stain EM and X-ray crystallography identified the variable region V of the NFL trimer to be the common ... More
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Less
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type secretion system T SS and target leukocytes to subvert host defenses T SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T SS and drives secretion via a Brownian ratchet mechanism Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin an RTX leukotoxin essential for B pertussis colonization have been shown to target the RTX domain and prevent binding to the M integrin receptor Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform ... More
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines. Less
The macrophage migration inhibitory factor MIF family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism s Previously we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel Despite structural and functional similarity the MIF homolog D-dopachrome tautomerase also called MIF- has low sequence identity prompting the question of whether this dynamic regulatory network is conserved Here we establish the structural basis of an allosteric site in MIF- showing with solution NMR that dynamic communication is preserved in MIF- despite differences in the primary sequence X-ray crystallography ... More
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily. Less
Toll-like receptors TLRs play an important role in the innate immune response While a lot is known about the structures of their extracellular parts many questions are still left unanswered when the structural basis of TLR activation is analyzed for the TLR intracellular domains Here we report the structure and dynamics of TLR toll-interleukin like TIR cytoplasmic domain in crystal and in solution We found that the TLR -TIR domain is capable of specific binding of Zn with nanomolar affinity Interactions with Zn are mediated by cysteine residues and and C is essential for the Zn binding Potential structures of ... More
Toll-like receptors (TLRs) play an important role in the innate immune response. While a lot is known about the structures of their extracellular parts, many questions are still left unanswered, when the structural basis of TLR activation is analyzed for the TLR intracellular domains. Here we report the structure and dynamics of TLR1 toll-interleukin like (TIR) cytoplasmic domain in crystal and in solution. We found that the TLR1-TIR domain is capable of specific binding of Zn with nanomolar affinity. Interactions with Zn are mediated by cysteine residues 667 and 686 and C667 is essential for the Zn binding. Potential structures of the TLR1-TIR/Zn complex were predicted in silico. Using the functional assays for the heterodimeric TLR1/2 receptor, we found that both Zn addition and Zn depletion affect the activity of TLR1, and C667A mutation disrupts the receptor activity. Analysis of C667 position in the TLR1 structure and possible effects of C667A mutation, suggests that zinc-binding ability of TLR1-TIR domain is critical for the receptor activation. Less
The -lactamase of Mycobacterium tuberculosis BlaC is susceptible to inhibition by clavulanic acid The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli The variant that was found to confer the most inhibitor resistance K R as well as variant G N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance NMR relaxation experiments to probe structural and dynamic properties The G N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca s The conformational change ... More
The β-lactamase of Mycobacterium tuberculosis, BlaC, is susceptible to inhibition by clavulanic acid. The ability of this enzyme to escape inhibition through mutation was probed using error-prone PCR combined with functional screening in Escherichia coli. The variant that was found to confer the most inhibitor resistance, K234R, as well as variant G132N that was found previously were characterized using X-ray crystallography and nuclear magnetic resonance (NMR) relaxation experiments to probe structural and dynamic properties. The G132N mutant exists in solution in two almost equally populated conformations that exchange with a rate of ca. 88 s−1. The conformational change affects a broad region of the enzyme. The crystal structure reveals that the Asn132 side chain forces the peptide bond between Ser104 and Ile105 in a cis-conformation. The crystal structure suggests multiple conformations for several side chains (e.g., Ser104 and Ser130) and a short loop (positions 214 to 216). In the K234R mutant, the active-site dynamics are significantly diminished with respect to the wild-type enzyme. These results show that multiple evolutionary routes are available to increase inhibitor resistance in BlaC and that active-site dynamics on the millisecond time scale are not required for catalytic function. Less
The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics The -lactamase BlaC of this pathogen prevents the use of -lactam antibiotics except in combination with a -lactamase inhibitor To understand if exposure to such inhibitors can easily result in resistance a BlaC evolution experiment was performed studying the evolutionary adaptability against the inhibitor sulbactam Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam The G S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for ... More
The current rise of antibiotic resistant forms of Mycobacterium tuberculosis is a global health threat that calls for new antibiotics. The β-lactamase BlaC of this pathogen prevents the use of β-lactam antibiotics, except in combination with a β-lactamase inhibitor. To understand if exposure to such inhibitors can easily result in resistance, a BlaC evolution experiment was performed, studying the evolutionary adaptability against the inhibitor sulbactam. Several amino acid substitutions in BlaC were shown to confer reduced sensitivity to sulbactam. The G132S mutation causes a reduction in the rate of nitrocefin and ampicillin hydrolysis and simultaneously reduces the sensitivity for sulbactam inhibition. Introduction of the side chain moiety of Ser132 causes the 104–105 peptide bond to assume the cis conformation and the side chain of Ser104 to be rotated toward the sulbactam adduct with which it forms a hydrogen bond not present in the wild-type enzyme. The gatekeeper residue Ile105 also moves. These changes in the entrance of the active site can explain the decreased affinity of G132S BlaC for both substrates and sulbactam. Our results show that BlaC can easily acquire a reduced sensitivity for sulbactam, with a single-amino acid mutation, which could hinder the use of combination therapies. Less
Eukaryotic proliferating cell nuclear antigen PCNA plays an essential role in orchestrating the assembly of the replisome complex stimulating processive DNA synthesis and recruiting other regulatory proteins during the DNA damage response PCNA and its binding partner network are relatively conserved in eukaryotes and it exhibits extraordinary structural similarity across species However despite this structural similarity the PCNA of a given species is rarely functional in heterologous systems In this report we determined the X-ray crystal structure of Neurospora crassa PCNA NcPCNA and compared its structure function relationship with other available PCNA studies to understand this cross-species incompatibility We found ... More
Eukaryotic proliferating cell nuclear antigen (PCNA) plays an essential role in orchestrating the assembly of the replisome complex, stimulating processive DNA synthesis, and recruiting other regulatory proteins during the DNA damage response. PCNA and its binding partner network are relatively conserved in eukaryotes, and it exhibits extraordinary structural similarity across species. However, despite this structural similarity, the PCNA of a given species is rarely functional in heterologous systems. In this report, we determined the X-ray crystal structure of Neurospora crassa PCNA (NcPCNA) and compared its structure–function relationship with other available PCNA studies to understand this cross-species incompatibility. We found two regions, the interdomain connecting loop (IDCL) and J loop structures, vary significantly among PCNAs. In particular, the J loop deviates in NcPCNA from that in Saccharomyces cerevisiae PCNA (ScPCNA) by 7 Å. Differences in the IDCL structures result in varied binding affinities of PCNAs for the subunit Pol32 of DNA polymerase delta and for T2-amino alcohol, a small-molecule inhibitor of human PCNA. To validate that these structural differences are accountable for functional incompatibility in S. cerevisiae, we generated NcPCNA mutants mimicking IDCL and J loop structures of ScPCNA. Our genetic analyses suggested that NcPCNA mutants are fully functional in S. cerevisiae. The susceptibility of the strains harboring ScPCNA mimics of NcPCNA to various genotoxic agents was similar to that in yeast cells expressing ScPCNA. Taken together, we conclude that in addition to the overall architecture of PCNA, structures of the IDCL and J loop of PCNA are critical determinants of interspecies functional compatibility. Less
A series of nondeuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized and found to potently inhibit severe acute respiratory syndrome coronavirus- CL protease in biochemical and cell-based assays Several of the inhibitors were also found to be nanomolar inhibitors of Middle East respiratory syndrome coronavirus CL protease The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding The spatial disposition of the compounds disclosed herein provides ... More
A series of nondeuterated and deuterated dipeptidyl aldehyde and masked aldehyde inhibitors that incorporate in their structure a conformationally constrained cyclohexane moiety was synthesized and found to potently inhibit severe acute respiratory syndrome coronavirus-2 3CL protease in biochemical and cell-based assays. Several of the inhibitors were also found to be nanomolar inhibitors of Middle East respiratory syndrome coronavirus 3CL protease. The corresponding latent aldehyde bisulfite adducts were found to be equipotent to the precursor aldehydes. High-resolution cocrystal structures confirmed the mechanism of action and illuminated the structural determinants involved in binding. The spatial disposition of the compounds disclosed herein provides an effective means of accessing new chemical space and optimizing pharmacological activity. The cellular permeability of the identified inhibitors and lack of cytotoxicity warrant their advancement as potential therapeutics for COVID-19. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- infection continues to be a serious global public health threat The C-like protease CLpro is a virus protease encoded by SARS-CoV- which is essential for virus replication We have previously reported a series of small-molecule CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV- in cell culture and in animal models Here we generated a series of deuterated variants of a CLpro inhibitor GC and evaluated the antiviral effect against SARS-CoV- The deuterated GC displayed potent inhibitory activity against SARS-CoV- in the enzyme- and the cell-based assays The K -hACE mice develop ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2–treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2. Less
Rhodopsins most of which are proton pumps generating transmembrane electrochemical proton gradients span all three domains of life are abundant in the biosphere and could play a crucial role in the early evolution of life on earth Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins rhodopsins from unicellular eukaryotes have not been well characterized To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach we performed a structural and functional analysis of the light-driven proton pump LR Mac from the ... More
Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor. Less
SARS-CoV- is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades The development of a universal human coronavirus vaccine could prevent future pandemics We characterize antibodies isolated from four COVID- subjects and identify SARS-CoV- neutralizing antibodies One targets the N-terminal domain NTD one recognizes an epitope in S and bind the receptor-binding domain RBD Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV- by effectively blocking binding of both the SARS-CoV- and SARS-CoV- RBDs to the ACE receptor Using the K -hACE transgenic mouse model we demonstrate that the neutralization potency and antibody ... More
SARS-CoV-2 is one of three coronaviruses that have crossed the animal-to-human barrier and caused widespread disease in the past two decades. The development of a universal human coronavirus vaccine could prevent future pandemics. We characterize 198 antibodies isolated from four COVID-19+ subjects and identify 14 SARS-CoV-2 neutralizing antibodies. One targets the N-terminal domain (NTD), one recognizes an epitope in S2, and 11 bind the receptor-binding domain (RBD). Three anti-RBD neutralizing antibodies cross-neutralize SARS-CoV-1 by effectively blocking binding of both the SARS-CoV-1 and SARS-CoV-2 RBDs to the ACE2 receptor. Using the K18-hACE transgenic mouse model, we demonstrate that the neutralization potency and antibody epitope specificity regulates the in vivo protective potential of anti-SARS-CoV-2 antibodies. All four cross-neutralizing antibodies neutralize the B.1.351 mutant strain. Thus, our study reveals that epitopes in S2 can serve as blueprints for the design of immunogens capable of eliciting cross-neutralizing coronavirus antibodies. Less
Human cannabinoid receptor CB plays an important role in the immune system and is an attractive therapeutic target for pain and for inflammatory and neurodegenerative diseases However the structural basis of CB agonist selectivity is still elusive Here we describe a detailed protocol for the determination of the crystal structure of antagonist AM -bound CB This methodology could be applied to the structural studies of CB with diverse antagonists and agonists or to other class A G-protein-coupled receptors
Sphingosine- -phosphate S P regulates numerous important physiological functions including immune response and vascular integrity via its cognate receptors S PR to S PR however it remains unclear how S P activates S PRs upon binding Here we determined the crystal structure of the active human S PR in complex with its natural agonist S P at - resolution S P exhibits an unbent conformation in the long tunnel which penetrates through the receptor obliquely Compared with the inactive S PR structure four residues surrounding the alkyl tail of S P the quartet core exhibit orchestrating rotamer changes that accommodate ... More
Sphingosine-1-phosphate (S1P) regulates numerous important physiological functions, including immune response and vascular integrity, via its cognate receptors (S1PR1 to S1PR5); however, it remains unclear how S1P activates S1PRs upon binding. Here, we determined the crystal structure of the active human S1PR3 in complex with its natural agonist S1P at 3.2-Å resolution. S1P exhibits an unbent conformation in the long tunnel, which penetrates through the receptor obliquely. Compared with the inactive S1PR1 structure, four residues surrounding the alkyl tail of S1P (the “quartet core”) exhibit orchestrating rotamer changes that accommodate the moiety, thereby inducing an active conformation. In addition, we reveal that the quartet core determines G protein selectivity of S1PR3. These results offer insight into the structural basis of activation and biased signaling in G protein–coupled receptors and will help the design of biased ligands for optimized therapeutics. Less
The Parkin co-regulated gene protein PACRG binds at the inner junction between doublet microtubules of the axoneme a structure found in flagella and cilia PACRG binds to the adaptor protein meiosis expressed gene MEIG but how they bind to microtubules is unknown Here we report the crystal structure of human PACRG in complex with MEIG PACRG adopts a helical repeat fold with a loop that interacts with MEIG Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation ... More
The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less
The zinc-finger transcription factor Helios is critical for maintaining the identity anergic phenotype and suppressive activity of regulatory T Treg cells While it is an attractive target to enhance the efficacy of currently approved immunotherapies no existing approaches can directly modulate Helios activity or abundance Here we report the structure-guided development of small molecules that recruit the E ubiquitin ligase substrate receptor cereblon to Helios thereby promoting its degradation Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells establishing a route towards Helios-targeting therapeutics More generally this study provides a framework for the development ... More
The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity. Less
The leukotriene B receptor BLT regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious allergic metabolic and tumorigenic human diseases Here we present a crystal structure of human BLT hBLT in complex with a selective antagonist MK-D- developed for the treatment of type diabetes and other inflammatory conditions Comprehensive analysis of the structure and structure-activity relationship data reinforced by site-directed mutagenesis and docking studies reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species The structure helps to identify a putative membrane-buried ligand access channel ... More
The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design. Less
The adhesion G protein coupled receptor CD and its ligand complement decay-accelerating factor CD are important binding partners in the human immune system Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis as well as various cancers Previous literatures have indicated that the CD includes to epidermal growth factor EGF domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat SCR domains of CD However the details of this interaction remain elusive especially why the CD binds with the highest affinity to the shortest isoform ... More
The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors. Less
Human parainfluenza virus type III HPIV is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations including the immunocompromised There are currently no effective vaccines or therapeutics available resulting in tens of thousands of hospitalizations per year In an effort to discover a protective antibody against HPIV we screened the B cell repertoires from peripheral blood tonsils and spleen from healthy children and adults These analyses yielded five monoclonal antibodies that potently neutralized HPIV in vitro These HPIV -neutralizing antibodies targeted two non-overlapping epitopes of the HPIV F protein with most targeting the apex Prophylactic ... More
Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals. Less
Alginate is the structural polysaccharide of the cell wall of brown algae which is an important carbon source for marine life The depolymerization of alginate is dependent on alginate lyases Recent studies showed that the alginate utilization ability had been obtained by human gut microbes In contrast to the great number of studies on alginate lyases from marine soil organisms studies on alginate lyases from gut microbes are still limited Here the structure of a polysaccharide lyase family PL alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography which represents the cluster of two-domain PL alginate ... More
Alginate is the structural polysaccharide of the cell wall of brown algae, which is an important carbon source for marine life. The depolymerization of alginate is dependent on alginate lyases. Recent studies showed that the alginate utilization ability had been obtained by human gut microbes. In contrast to the great number of studies on alginate lyases from marine/soil organisms, studies on alginate lyases from gut microbes are still limited. Here, the structure of a polysaccharide lyase family 6 (PL6) alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography, which represents the cluster of two-domain PL6 alginate lyases from Bacteroidetes. Similar with the two-domain alginate lyase AlyGC originated from marine bacterium, both the N terminal domain (NTD) and C terminal domain (CTD) of BcAlyPL6 show right-handed parallel β-helix fold. However, unlike AlyGC, which forms a homodimer, BcAlyPL6 functions as a monomer. Biochemical analysis indicates that the substrate binding affinity is mainly contributed by the NTD while the CTD of BcAlyPL6 is involved in the formation of −1 subsite, which is essential for substrate turnover rate. Furthermore, CTD is involved in shaping a closed catalytic pocket, and deletion of it leads to increased activity towards highly polymerized substrate. Structure comparison of PL6 family alginate lyases implies that the linkers of two-domain alginate lyases might have evolutionary relationship with the N/C terminal extension of single-domain lyases. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
Light-oxygen-voltage LOV domains are widespread photosensory modules that can be used in fluorescence microscopy optogenetics and controlled production of reactive oxygen species All of the currently known LOV domains have absorption maxima in the range of to nm and it is not clear whether they can be shifted significantly using mutations Here we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP Gln to asparagine aspartate glutamate histidine lysine and arginine Absorption spectra of all of the mutants are blue-shifted with the maximal shift of nm ... More
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins. Less
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution yet the unique contributions of multimeric IgM antibodies to infection control are not well understood This is partially due to the difficulty of distinguishing low-affinity IgM secreted rapidly by plasmablasts from high-affinity antibodies derived from later-arising memory cells We developed a pipeline to express B cell receptors BCRs from Plasmodium falciparum specific IgM and IgG human memory B cells MBCs as both IgM and IgG molecules BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity Crystallization of one IgM MBC-derived antibody complexed with antigen defined a linear epitope within ... More
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines. Less
The Parkin co-regulated gene protein PACRG binds at the inner junction between doublet microtubules of the axoneme a structure found in flagella and cilia PACRG binds to the adaptor protein meiosis expressed gene MEIG but how they bind to microtubules is unknown Here we report the crystal structure of human PACRG in complex with MEIG PACRG adopts a helical repeat fold with a loop that interacts with MEIG Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation ... More
The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less
Mutations in two different domains of the ubiquitously expressed TRIM protein give rise to two clinically separate diseases one of which is Limb-girdle muscular dystrophy type H LGMD H Uncovering the muscle-specific role of TRIM in LGMD H pathogenesis has proven difficult as neurogenic phenotypes independent of LGMD H pathology are present in TRIM KO mice We previously established a platform to study LGMD H pathogenesis using Drosophila melanogaster as a model Here we show that LGMD H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM Furthermore transgenic expression of a subset ... More
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration. Less
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA Here we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides Kinetic NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension and in part because of the greater distance between the attacking RNA primer -hydroxyl and the phosphate of the incoming threo-nucleotide ... More
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3′-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands. Less
White spot syndrome virus WSSV the causative agent of white spot disease WSD severely affecting crustacean life forms is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target leading to various anti-cancer drugs The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase wTS Here we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate dUMP and methotrexate at a resolution of and ... More
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- and other SARS-like-CoVs encode tandem macrodomains within non-structural protein nsp The first macrodomain Mac is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose MAR from target proteins Mac likely counters host-mediated anti-viral ADP-ribosylation a posttranslational modification that is part of the host response to viral infections Mac is essential for pathogenesis in multiple animal models of CoV infection implicating it as a virulence factor and potential therapeutic target Here we report the crystal structure of SARS-CoV- Mac in complex with ADP-ribose SARS-CoV- SARS-CoV and MERS-CoV Mac exhibit similar structural folds and all proteins ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. Less
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork In eukaryotes proliferating cell nuclear antigen PCNA plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability PCNA from several organisms including Saccharomyces cerevisiae has been structurally characterised However the structural analyses of PCNA from fungal pathogens are limited Recently we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S cerevisiae Still it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic ... More
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
Electron crystallography of sub-micrometre-sized D protein crystals has emerged recently as a valuable field of structural biology In meso crystallization methods utilizing lipidic mesophases particularly lipidic cubic phases LCPs can produce high-quality D crystals of membrane proteins MPs A major step towards realizing D electron crystallography of MP crystals grown in meso is to demonstrate electron diffraction from such crystals The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals Additionally the crystals have to be thin enough to let electrons traverse them without significant multiple scattering In the present ... More
Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media. Less
Structural studies of challenging targets such as G protein-coupled receptors GPCRs have accelerated during the last several years due to the development of new approaches including small-wedge and serial crystallography Here we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase LCP grown microcrystals of two human GPCRs Cysteinyl leukotriene receptors and CysLT R and CysLT R in complex with various antagonists Five datasets were collected using small-wedge synchrotron crystallography SWSX at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions Two datasets were collected using X-ray free electron laser XFEL serial ... More
Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing. Less
Excitatory amino acid transporters EAATs harness Na K and H gradients for fast and efficient glutamate removal from the synaptic cleft Since each glutamate is cotransported with three Na ions Na gradients are the predominant driving force for glutamate uptake We combined all-atom molecular dynamics simulations fluorescence spectroscopy and x-ray crystallography to study Na substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type Na -only bound GltPh at - resolution revealed the fully open outward-facing state primed for subsequent substrate binding Simulations and kinetic experiments established that only the binding of two Na ... More
Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations. Less
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly Vertebrate transferrins coordinate iron through interactions with two tyrosines an aspartate a histidine and a carbonate anion and conformational changes that occur upon iron binding and release have been described Much less is known about the structure and functions of insect transferrin- Tsf which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf differs from that of the vertebrate transferrins Here we report the first crystal structure resolution of an ... More
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans. Less
Phytoplankton is the base of the marine food chain oxygen carbon cycle playing a global role in climate and ecology Nucleocytoplasmic Large DNA Viruses regulating the dynamics of phytoplankton comprise genes of rhodopsins of two distinct families We present a function-structure characterization of two homologous proteins representatives of family of viral rhodopsins OLPVR and VirChR VirChR is a highly selective Ca -dependent Na K - conducting channel and in contrast to known cation channelrhodopsins ChRs is impermeable to Ca ions In human neuroblastoma cells upon illumination VirChR depolarizes the cell membrane to a level sufficient to fire neurons It suggests ... More
Phytoplankton is the base of the marine food chain, oxygen, carbon cycle playing a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses regulating the dynamics of phytoplankton comprise genes of rhodopsins of two distinct families. We present a function-structure characterization of two homologous proteins representatives of family 1 of viral rhodopsins, OLPVR1 and VirChR1. VirChR1 is a highly selective, Ca2+-dependent, Na+/K+- conducting channel and, in contrast to known cation channelrhodopsins (ChRs), is impermeable to Ca2+ ions. In human neuroblastoma cells, upon illumination, VirChR1 depolarizes the cell membrane to a level sufficient to fire neurons. It suggests its unique optogenetic potential. 1.4 Å resolution structure of OLPVR1 reveals their remarkable difference from the known channelrhodopsins and a unique ion-conducting pathway. The data suggest that viral channelrhodopsins mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, their key role in global phytoplankton dynamics. Less
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan As lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes cross-linked to the matrix How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may ... More
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide–peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2,3,4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan As Lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a ... More
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
- - protein isoforms regulate multiple processes in eukaryotes including apoptosis and cell division - - proteins preferentially recognize phosphorylated unstructured motifs justifying the protein-peptide binding approach to study - - phosphotarget complexes Tethering of human - - with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides simultaneously facilitating purification and crystallization Nevertheless the broader applicability to other - - isoforms and phosphopeptides was unclear Here we designed a novel - - chimera with a conserved phosphopeptide from BAD whose complex with - - is a gatekeeper of apoptosis regulation The ... More
14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the “chimeric” approach for studies of other relevant 14-3-3 complexes. Less
SARS-CoV- is a betacoronavirus virus responsible for the COVID- pandemic Here we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody CV isolated from a patient infected with SARS-CoV- in complex with the receptor binding domain RBD The structure reveals CV s epitope overlaps with the human ACE receptor binding site thus providing the structural basis for its neutralization by preventing ACE binding
Cation-chloride cotransporters CCCs regulate the movement of chloride across membranes controlling physiological processes from cell volume maintenance to neuronal signaling Human CCCs are clinical targets for existing diuretics and potentially additional indications Here we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter Caenorhabditis elegans KCC- We observe a core a fold conserved among CCCs Using structure-based sequence alignment we analyze similarities and differences to the C-terminal domains of other CCC family members We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved suggesting ... More
Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core a/� fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation. Less
Ghrelin is a gastric peptide hormone with important physiological functions The unique feature of ghrelin is its Serine acyl-modification which is essential for ghrelin s activity However it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity To address these questions we solved the crystal structure of the ghrelin receptor bound to antagonist The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E and R A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap crevasse between the TM and TM bundles that is rich in ... More
Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin’s activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation. Less
Pathogenic coronaviruses are a major threat to global public health as exemplified by severe acute respiratory syndrome coronavirus SARS-CoV Middle East respiratory syndrome coronavirus MERS-CoV and the newly emerged SARS-CoV- the causative agent of coronavirus disease COVID- We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus C-like protease CLpro an enzyme essential for viral replication The optimized compounds were effective against several human coronaviruses including MERS-CoV SARS-CoV and SARS-CoV- in an enzyme assay and in cell-based assays using Huh- and Vero E cell lines Two selected compounds showed antiviral effects against SARS-CoV- in cultured primary ... More
Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. Less
G-protein-coupled receptors GPCRs the largest family of cell-surface membrane proteins mediate the intracellular signal transduction of many external ligands Thus GPCRs have become important drug targets X-ray crystal structures of GPCRs are very useful for structure-based drug design SBDD Herein we produced a new antibody SRP targeting the thermostabilised apocytochrome b from Escherichia coli M W H I R L BRIL We found that a fragment of this antibody SRP Fab facilitated the crystallisation of the BRIL-tagged ligand bound GPCRs HT B and AT R Furthermore the electron densities of the ligands were resolved suggesting that SPR Fab is versatile ... More
G-protein-coupled receptors (GPCRs)�the largest family of cell-surface membrane proteins�mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them. Less
Emergent nanoscale order in materials such as self-assembled lipid phases colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering SAXS Frequently residual disorder in these materials prevents high-resolution D structural characterization Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local D structure via a rich real-space distribution of three- and four-body statistics We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid revealing non-uniform curvature in the lipid channels likely due to phase separation of the lipids in the membrane Our many-body technique has general ... More
Emergent nanoscale order in materials such as self-assembled lipid phases, colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering (SAXS). Frequently, residual disorder in these materials prevents high-resolution 3D structural characterization. Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local 3D structure via a rich, real-space distribution of three- and four-body statistics. We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid, revealing non-uniform curvature in the lipid channels, likely due to phase separation of the lipids in the membrane. Our many-body technique has general applicability to nanomaterials with order in the range 10 nm−1 μm currently targeted by synchrotron SAXS and has the potential to impact diverse research areas within chemistry, biology and materials science. Less
Chemokines and their receptors mediate cell migration which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor chemokine recognition less is known about endogenous chemokine-induced receptor activation and G-protein coupling Here we present the cryo-electron microscopy structures of interleukin- IL- also known as CXCL -activated human CXC chemokine receptor CXCR in complex with Gi protein along with a crystal structure of CXCR bound to a designed allosteric antagonist Our results reveal a unique shallow mode of binding between CXCL and ... More
Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor–chemokine recognition2,3,4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles. Less
The enzyme pyridoxal kinase PdxK catalyzes the conversion of pyridoxal to pyridoxal- -phosphate PLP using ATP as the co-factor The product pyridoxal- -phosphate plays a key role in several biological processes such as transamination decarboxylation and deamination In the present study full-length ORF of PdxK from Leishmania donovani LdPdxK was cloned and then purified using affinity chromatography LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH Biochemical analysis of LdPdxK with pyridoxal pyridoxamine pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK ... More
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5′-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5′-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors. Less
Of the eighteen hemagglutinin HA subtypes H H that have been identified in bats and aquatic birds many HA subtypes have been structurally characterized However several subtypes H H and H still require characterization To better understand all of these HA subtypes at the molecular level HA structures from an A H N A swine Missouri A an A H N A turkey Ontario an A H N A duck Memphis an A H N A mallard Gurjev and an A H N A wedge-tailed shearwater Western Australia were determined by X-ray crystallography at and resolution respectively The interactions between ... More
Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. Less
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea They possess highly diverse biological functions Explosion of research on microbial rhodopsins led to breakthroughs in their applications in particular in neuroscience An unexpected new discovery was a Na -pumping KR rhodopsin from Krokinobacter eikastus the first light-driven non-proton cation pump A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and therefore Na pumping cannot be understood in the framework ... More
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics. Less
Membrane-embedded sensor histidine kinases HKs and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment and are often crucial for their survival and pathogenicity The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms and regulate the activity of the cognate response regulator proteins with which they form two-component signaling systems TCSs Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed dubbed swinging piston helical rotation and diagonal scissoring Yet despite decades of studies there is no consensus on whether ... More
Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well. Less
Light-oxygen-voltage LOV domains are ubiquitous photosensory modules found in proteins from bacteria archaea and eukaryotes Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics with improved versions being continuously developed Many of the engineering efforts focused on the thermal stabilization of LOV domains Recently we described a naturally thermostable LOV domain from Chloroflexus aggregans Here we show that the discovered protein can be further stabilized using proline substitution We tested the effects of three mutations and found that the melting temperature of the A P mutant is raised by approximately C whereas mutations A P ... More
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. Less
Perovskites have seen significant research interest in the last decade As ternary and quaternary compounds their chemical space is exceptionally large yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error Here we report a high-throughput experimental framework for the discovery of new perovskite single crystals We use machine learning ML to guide the sequence of ever-improved robotic synthetic trials We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition We then use an ML ... More
Perovskites have seen significant research interest in the last decade. As ternary and quaternary compounds, their chemical space is exceptionally large, yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error. Here, we report a high-throughput experimental framework for the discovery of new perovskite single crystals. We use machine learning (ML) to guide the sequence of ever-improved robotic synthetic trials. We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition. We then use an ML model to predict the optimal conditions for the synthesis of a new perovskite single crystal, enabling us to report the first synthesis of (3-PLA)2PbCl4.This material exhibits strong blue emission, illustrating the applicability of the method in identifying new optoelectronic materials. Less
Glucagon-like peptide- receptor GLP- R is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type diabetes Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides however little is known about their extracellular domain ECD conformations in the absence of orthosteric ligands which has limited our understanding of their activation mechanism Here we report the resolution peptide-free crystal structure of the full-length human GLP- R in an inactive state which reveals a unique closed conformation of the ECD Disulfide cross-linking validates the physiological relevance of the ... More
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family. Less
Two-component signaling systems TCSs are a large and important class of sensory systems in bacteria archaea and some eukaryotes yet their mechanism of action is still not fully understood from the structural point of view Many TCS receptors are elongated flexible proteins with transmembrane TM regions and are difficult to work with Consequently truncated fragments of the receptors are often used in structural studies However it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins Recently we crystallized a fragment of Escherichia coli nitrate nitrite sensor histidine ... More
Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs has recently been discovered Unlike in the known rhodopsins in HeRs the N termini face the cytoplasm The function of HeRs remains unknown We present the structures of the bacterial HeR- C in two states at the resolution of which highlight its remarkable difference from all known rhodopsins The interior of HeR s extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity Schiff base cavity SBC surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes. Less
Human muscarinic receptor M belongs to the class A subfamily of the G-protein-coupled receptors GPCRs M has emerged as an attractive drug target for the treatment of Alzheimer s disease and schizophrenia Recent results showed that M -mediated cholinergic transmission is related to motor symptoms in Parkinson s disease Selective ligand design for the five muscarinic acetylcholine receptor mAchR subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets In order to obtain M -selective antagonists a new approach was tried to lock M into an inactive form by rationally designing an N ... More
Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer’s disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson’s disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination. Less
GPR is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington s disease and several psychiatric disorders Pathological malfunction of GPR signalling occurs primarily through the heterotrimeric Gs protein but it is unclear how GPR and Gs couple for signal transduction and whether a native ligand or other activating input is required Here we present the high-resolution structures of human GPR in three states a ligand-free state a Gs-coupled self-activation state and a potential allosteric ligand-bound state Together our structures reveal that extracellular loop occupies the ... More
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington’s disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52. Less
Phospholipase D enzymes PLDs are ubiquitous phosphodiesterases that produce phosphatidic acid PA a key second messenger and biosynthetic building block Although an orthologous bacterial Streptomyces sp strain PMF PLD structure was solved two decades ago the molecular basis underlying the functions of the human PLD enzymes hPLD remained unclear based on this structure due to the low homology between these sequences Here we describe the first crystal structures of hPLD and hPLD catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes Furthermore structure-based mutation studies and structures of inhibitor hPLD complexes allowed us ... More
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor–hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs was recently discovered In opposite to the known rhodopsins their N-termini face the cytoplasm HeRs structure and function remain unknown We present structures of two HeR- C states at showing its remarkable difference from all known rhodopsins Its internal extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity active site surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton acceptor from the Schiff base At acidic pH a planar triangle molecule acetate is present in ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes. Less
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB and CB Their high sequence similarity low agonist selectivity and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications Importantly missing structural information has significantly held back the development of promising CB -selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB Here we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB and CB in complex with Gi as well as agonist-bound CB crystal structure Of important scientific and therapeutic benefit our results reveal a ... More
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role. Less
Shiga toxin Stx is the major virulence factor of Shiga toxin-producing Escherichia coli STEC Stx evolves rapidly and as such new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies A new subtype Stx k was recently identified in E coli isolated from a wide range of sources including diarrheal patients animals and raw meats and was poorly detected by existing immunoassays In this study the structure of Stx kE Q was determined at resolution and the conservation of structure with Stx a was revealed A novel polyclonal antibody capable of neutralizing Stx k ... More
Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs. Less
Membrane integral ATP synthases produce adenosine triphosphate the universal energy currency of most organisms However important details of proton driven energy conversion are still unknown We present the first high-resolution structure of the in meso crystallized c-ring of subunits from spinach chloroplasts The structure reveals molecular mechanisms of intersubunit contacts in the c -ring and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane Similar densities were found in all known high-resolution structures of c-rings of F FO ATP synthases from archaea and bacteria to eukaryotes The densities might originate from isoprenoid quinones ... More
Membrane integral ATP synthases produce adenosine triphosphate, the universal “energy currency” of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it. Less
Cysteinyl leukotriene G protein-coupled receptors CysLT and CysLT regulate pro-inflammatory responses associated with allergic disorders While selective inhibition of CysLT R has been used for treating asthma and associated diseases for over two decades CysLT R has recently started to emerge as a potential drug target against atopic asthma brain injury and central nervous system disorders as well as several types of cancer Here we describe four crystal structures of CysLT R in complex with three dual CysLT R CysLT R antagonists The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular ... More
Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT2R in complex with three dual CysLT1R/CysLT2R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants. Less
Broadly HIV- neutralizing VRC class antibodies target the CD -binding site of Env They are derived from VH - antibody heavy chains paired with rare light chains expressing -amino acid-long CDRL s They have been isolated from infected subjects but have not yet been elicited by immunization Env-derived immunogens capable of binding the germline forms of VRC B cell receptors on naive B cells have been designed and evaluated in knockin mice However the elicited antibodies cannot bypass glycans present on the conserved position N of Env which restricts access to the CD -binding site Efforts to guide the appropriate ... More
Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2*02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies. Less
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding and understanding this diversity is key for designing selective or biased drugs for them Here we determine two crystal structures of the a A adrenergic receptor a AAR in complex with a partial agonist and an antagonist Key non-conserved residues from the ligand-binding pocket Phe and Tyr to G-protein coupling region Ile and Lys are discovered to play a key role in the interplay between partial agonism and biased signaling of a AAR which provides insights into the diversity of ligand ... More
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the a2A adrenergic receptor (a2AAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe7.39 and Tyr6.55) to G-protein coupling region (Ile34.51 and Lys34.56) are discovered to play a key role in the interplay between partial agonism and biased signaling of a2AAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors. Less
Adrenergic G-protein-coupled receptors GPCRs mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions Extensive studies have been carried out to investigate the structure and function of adrenergic receptors ARs However the structure of a adrenergic receptors aARs remains to be determined Here we report the structure of the human a C adrenergic receptor a CAR with the non-selective antagonist RS at Our structure mutations modeling and functional experiments indicate that a a CAR-specific D ECL -R ECL -Y network plays a role in determining a adrenergic subtype selectivity ... More
Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of � adrenergic receptors (�ARs). However, the structure of a adrenergic receptors (aARs) remains to be determined. Here, we report the structure of the human a2C adrenergic receptor (a2CAR) with the non-selective antagonist, RS79948, at 2.8 �. Our structure, mutations, modeling, and functional experiments indicate that a a2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining a2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in a2CAR is involved in receptor activation. Together, our structure of human a2CAR-RS79948 provides key insight into the mechanism underlying the a2 adrenergic receptor activation and subtype selectivity. Less
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error Perovskites are an example of materials having exceptional optoelectronic properties but require improvement with respect to stability and toxicity as they approach commercialization Exploring new types of perovskites is key to achieving these goals In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials This pipeline incorporates image recognition that detects crystals via convolutional neural networks with accuracy and uses parameter exploration to predict an optimal material with experimental data With this framework I discovered a new type ... More
Reaching the full potential of optoelectronic materials is often hindered by the years of necessary trial-and-error. Perovskites are an example of materials having exceptional optoelectronic properties, but require improvement with respect to stability and toxicity as they approach commercialization. Exploring new types of perovskites is key to achieving these goals. In this thesis I develop an accelerated materials discovery pipeline aimed at discovering new perovskite materials. This pipeline incorporates image recognition that detects crystals via convolutional neural networks with 95% accuracy and uses parameter exploration to predict an optimal material with experimental data. With this framework, I discovered a new type of perovskite single crystal, (3-PLA)2PbCl4, that employs a new ligand, 3-PLA, offering avenues to higher efficiency and more stable devices. This work develops a framework for discovering and optimizing materials in a wide chemical space and provides the groundwork for identifying new materials that lie beyond known chemical spaces. Less
Selective activation of the -opioid receptor DOP has great potential for the treatment of chronic pain benefitting from ancillary anxiolytic and antidepressant-like effects Moreover DOP agonists show reduced adverse effects as compared to -opioid receptor MOP agonists that are in the spotlight of the current opioid crisis Here we report the first crystal structures of the DOP in an activated state in complex with two relevant and structurally diverse agonists the potent opioid agonist peptide KGCHM and the small-molecule agonist DPI- at and resolution respectively Our study identifies key determinants for agonist recognition receptor activation and DOP selectivity revealing crucial ... More
Selective activation of the δ-opioid receptor (DOP) has great potential for the treatment of chronic pain, benefitting from ancillary anxiolytic and antidepressant-like effects. Moreover, DOP agonists show reduced adverse effects as compared to μ-opioid receptor (MOP) agonists that are in the spotlight of the current “opioid crisis.” Here, we report the first crystal structures of the DOP in an activated state, in complex with two relevant and structurally diverse agonists: the potent opioid agonist peptide KGCHM07 and the small-molecule agonist DPI-287 at 2.8 and 3.3 Å resolution, respectively. Our study identifies key determinants for agonist recognition, receptor activation, and DOP selectivity, revealing crucial differences between both agonist scaffolds. Our findings provide the first investigation into atomic-scale agonist binding at the DOP, supported by site-directed mutagenesis and pharmacological characterization. These structures will underpin the future structure-based development of DOP agonists for an improved pain treatment with fewer adverse effects. Less
The release of synthetic chemical pollutants in the environment is posing serious health risks Enzymes including oxygenases play a crucial role in xenobiotic degradation In the present study we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants Fosmid clones possessing dioxygenase activity were further sequenced and their genes were identified using bioinformatics tools Two positive fosmid clones SD and RW suggested the presence of -dihydroxybiphenyl -dioxygenase BphC-SD and catechol -dioxygenase C O-RW respectively Recombinant versions of these enzymes were purified ... More
The release of synthetic chemical pollutants in the environment is posing serious health risks. Enzymes, including oxygenases, play a crucial role in xenobiotic degradation. In the present study, we employed a functional metagenomics approach to overcome the limitation of cultivability of microbes under standard laboratory conditions in order to isolate novel dioxygenases capable of degrading recalcitrant pollutants. Fosmid clones possessing dioxygenase activity were further sequenced, and their genes were identified using bioinformatics tools. Two positive fosmid clones, SD3 and RW1, suggested the presence of 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC-SD3) and catechol 2,3-dioxygenase (C23O-RW1), respectively. Recombinant versions of these enzymes were purified to examine their pollutant-degrading abilities. The crystal structure of BphC-SD3 was determined at 2.6-Å resolution, revealing a two-domain architecture, i.e., N-terminal and C-terminal domains, with the sequential arrangement of βαβββ in each domain, characteristic of Fe-dependent class II type I extradiol dioxygenases. The structure also reveals the presence of conserved amino acids lining the catalytic pocket and Fe3+ metal ion in the large funnel-shaped active site in the C-terminal domain. Further studies suggest that Fe3+ bound in the BphC-SD3 active site probably imparts aerobic stability. We further demonstrate the potential application of BphC-SD3 in biosensing of catecholic compounds. The halotolerant and oxygen-resistant properties of these enzymes reported in this study make them potential candidates for bioremediation and biosensing applications. Less
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens SAgs which cross-link major histocompatibility complex class II MHC II molecule and T-cell receptor TCR to stimulate large numbers of T cells at extremely low concentrations SAgs are associated with food poisoning and toxic shock syndrome To date genetically distinct staphylococcal SAgs have been reported This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N SEN SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal -grasp domain Amino acid and structure alignments revealed that several critical ... More
Staphylococcus aureus strains produce a unique family of immunostimulatory exotoxins termed as bacterial superantigens (SAgs), which cross-link major histocompatibility complex class II (MHC II) molecule and T-cell receptor (TCR) to stimulate large numbers of T cells at extremely low concentrations. SAgs are associated with food poisoning and toxic shock syndrome. To date, 26 genetically distinct staphylococcal SAgs have been reported. This study reports the first X-ray structure of newly characterized staphylococcal enterotoxin N (SEN). SEN possesses the classical two domain architecture that includes an N-terminal oligonucleotide-binding fold and a C-terminal β-grasp domain. Amino acid and structure alignments revealed that several critical amino acids that are proposed to be responsible for MHC II and TCR molecule engagements are variable in SEN, suggesting that SEN may adopt a different binding mode to its cellular receptors. This work helps better understand the mechanisms of action of SAgs. Less
The elicitation of broadly neutralizing antibodies bNAbs against the HIV- envelope glycoprotein Env trimer remains a major vaccine challenge Most cross-conserved protein determinants are occluded by self-N-glycan shielding limiting B cell recognition of the underlying polypeptide surface The exceptions to the contiguous glycan shield include the conserved receptor CD binding site CD bs and glycoprotein gp elements proximal to the furin cleavage site Accordingly we performed heterologous trimer-liposome prime boosting in rabbits to drive B cells specific for cross-conserved sites To preferentially expose the CD bs to B cells we eliminated proximal N-glycans while maintaining the native-like state of the ... More
The elicitation of broadly neutralizing antibodies (bNAbs) against the HIV-1 envelope glycoprotein (Env) trimer remains a major vaccine challenge. Most cross-conserved protein determinants are occluded by self-N-glycan shielding, limiting B cell recognition of the underlying polypeptide surface. The exceptions to the contiguous glycan shield include the conserved receptor CD4 binding site (CD4bs) and glycoprotein (gp)41 elements proximal to the furin cleavage site. Accordingly, we performed heterologous trimer-liposome prime:boosting in rabbits to drive B cells specific for cross-conserved sites. To preferentially expose the CD4bs to B cells, we eliminated proximal N-glycans while maintaining the native-like state of the cleavage-independent NFL trimers, followed by gradual N-glycan restoration coupled with heterologous boosting. This approach successfully elicited CD4bs-directed, cross-neutralizing Abs, including one targeting a unique glycan-protein epitope and a bNAb (87% breadth) directed to the gp120:gp41 interface, both resolved by high-resolution cryoelectron microscopy. This study provides proof-of-principle immunogenicity toward eliciting bNAbs by vaccination Less
Well-ordered HIV- envelope glycoprotein Env trimers are prioritized for clinical evaluation and there is a need for an improved understanding about how elicited B cell responses evolve following immunization To accomplish this we prime-boosted rhesus macaques with clade C NFL trimers and identified unique Ab lineages from single-sorted Env-specific memory B cells We traced all lineages in high-throughput heavy chain HC repertoire Rep-seq data generated from multiple immune compartments and time points and expressed several as monoclonal Abs mAbs Our results revealed broad dissemination and high levels of somatic hypermutation SHM of most lineages including tier virus neutralizing lineages following ... More
Well-ordered HIV-1 envelope glycoprotein (Env) trimers are prioritized for clinical evaluation, and there is a need for an improved understanding about how elicited B cell responses evolve following immunization. To accomplish this, we prime-boosted rhesus macaques with clade C NFL trimers and identified 180 unique Ab lineages from ∼1,000 single-sorted Env-specific memory B cells. We traced all lineages in high-throughput heavy chain (HC) repertoire (Rep-seq) data generated from multiple immune compartments and time points and expressed several as monoclonal Abs (mAbs). Our results revealed broad dissemination and high levels of somatic hypermutation (SHM) of most lineages, including tier 2 virus neutralizing lineages, following boosting. SHM was highest in the Ab complementarity determining regions (CDRs) but also surprisingly high in the framework regions (FRs), especially FR3. Our results demonstrate the capacity of the immune system to affinity-mature large numbers of Env-specific B cell lineages simultaneously, supporting the use of regimens consisting of repeated boosts to improve each Ab, even those belonging to less expanded lineages. Less
The investigational drugs E indisulam and tasisulam aryl-sulfonamides promote the degradation of the splicing factor RBM in a proteasome-dependent mechanism While the activity critically depends on the Cullin RING ligase substrate receptor DCAF the molecular details remain elusive Here we present the cryo-EM structure of the DDB -DCAF -DDA core ligase complex bound to RBM and E at resolution together with crystal structures of engineered subcomplexes We show that DCAF adopts a novel fold stabilized by DDA and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF Our data demonstrates how aryl-sulfonamides ... More
The investigational drugs E7820, indisulam and tasisulam (aryl-sulfonamides) promote the degradation of the splicing factor RBM39 in a proteasome-dependent mechanism. While the activity critically depends on the Cullin RING ligase substrate receptor DCAF15, the molecular details remain elusive. Here we present the cryo-EM structure of the DDB1-DCAF15-DDA1 core ligase complex bound to RBM39 and E7820 at 4.4 Å resolution, together with crystal structures of engineered subcomplexes. We show that DCAF15 adopts a novel fold stabilized by DDA1, and that extensive protein-protein contacts between the ligase and substrate mitigate low affinity interactions between aryl-sulfonamides and DCAF15. Our data demonstrates how aryl-sulfonamides neo-functionalize a shallow, non-conserved pocket on DCAF15 to selectively bind and degrade RBM39 and the closely related splicing factor RBM23 without the requirement for a high affinity ligand, which has broad implications for the de novo discovery of molecular glue degraders. Less
The class B family of G-protein-coupled receptors GPCRs has long been a paradigm for peptide hormone recognition and signal transduction One class B GPCR the glucagon-like peptide- receptor GLP- R has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease The previously determined structures of inactive GLP- R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of designed mutations Here we systematically summarize all mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical ... More
The class B family of G-protein-coupled receptors (GPCRs) has long been a paradigm for peptide hormone recognition and signal transduction. One class B GPCR, the glucagon-like peptide-1 receptor (GLP-1R), has been considered as an anti-diabetes drug target and there are several peptidic drugs available for the treatment of this overwhelming disease. The previously determined structures of inactive GLP-1R in complex with two negative allosteric modulators include ten thermal-stabilizing mutations that were selected from a total of 98 designed mutations. Here we systematically summarize all 98 mutations we have tested and the results suggest that the mutagenesis strategy that strengthens inter-helical hydrophobic interactions shows the highest success rate. We further investigate four back mutations by thermal-shift assay, crystallization and molecular dynamic simulations, and conclude that mutation I1962.66bF increases thermal stability intrinsically and that mutation S2714.47bA decreases crystal packing entropy extrinsically, while mutations S1932.63bC and M2333.36bC may be dispensable since these two cysteines are not disulfide-linked. Our results indicate intrinsic connections between different regions of GPCR transmembrane helices and the current data suggest a general mutagenesis principle for structural determination of GPCRs and other membrane proteins. Less
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase polyketide synthase NRPS PKS followed by a number of tailoring' steps to form the two ring systems that are present in the mature product It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels Alder reaction Here the crystal structure of a selenomethionine-labelled truncated form of IdmH IdmH- was solved using single-wavelength anomalous dispersion SAD phasing This truncated variant allows consistent and easy crystallization but importantly the structure was used as a search model in molecular replacement allowing the full-length ... More
Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of `tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels–Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99–107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7 Å resolution. IdmH is a homodimer, with the individual protomers consisting of an α+β barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels–Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels–Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399. Less
Recently two groups of rhodopsin genes were identified in large double-stranded DNA viruses The structure and function of viral rhodopsins are unknown We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II OLPVRII of group It forms a pentamer with a symmetrical bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of arginines whereas phenylalanines form a hydrophobic barrier in its exit The proton donor E is placed in the helix B The structure is unique among the known rhodopsins Structural and functional data and molecular dynamics suggest that OLPVRII ... More
Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize the structure and function of Mesocricetus auratus ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize the structure and function of Mesocricetus auratus MIF (MaMIF) and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant MaMIF was cloned, expressed and purified in a bacterial expression system. The MaMIF primary sequence, biochemical properties, and crystal structure analysis showed greater similarity with human MIF. The crystal structure of MaMIF illustrates that it forms a homotrimer as known in human and mouse. However, MaMIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that MaMIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of MaMIF into HapT1 pancreatic tumor-bearing hamsters significantly enhanced the tumor growth and tumor-associated angiogenesis. Together, the current study shows a structural and functional similarity between the hamster and human MIF. Moreover, it has demonstrated that a high level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Macrophage migration inhibitory factor MIF is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions the information obtained from these models is biased towards a specific species In experimental science results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans Syrian golden hamster Mesocricetus auratus is a clinically relevant animal model for multiple human diseases Hence the major objectives of this study were to characterize structure and function of hamster MIF and ... More
Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that increasingly is being studied in cancers and inflammatory diseases. Though murine models have been instrumental in understanding the functional role of MIF in different pathological conditions, the information obtained from these models is biased towards a specific species. In experimental science, results obtained from multiple clinically relevant animal models always provide convincing data that might recapitulate in humans. Syrian golden hamster (Mesocricetus auratus), is a clinically relevant animal model for multiple human diseases. Hence, the major objectives of this study were to characterize structure and function of hamster MIF, and finally evaluate its effect on pancreatic tumor growth in vivo. Initially, the recombinant hamster MIF (rha-MIF) was cloned, expressed and purified in bacterial expression system. The rha-MIF primary sequence, biochemical properties and crystal structure analysis showed a greater similarity with human MIF. The crystal structure of hamster MIF illustrates that it forms a homotrimer as known in human and mouse. However, hamster MIF exhibits some minor structural variations when compared to human and mouse MIF. The in vitro functional studies show that rha-MIF has tautomerase activity and enhances activation and migration of hamster peripheral blood mononuclear cells (PBMCs). Interestingly, injection of rha-MIF into HapT1 pancreatic tumor bearing hamsters significantly enhanced the tumor growth and tumor associated angiogenesis. Together, the current study shows a structural and functional similarity between hamster and human MIF. Moreover, it has demonstrated that a high-level of circulating MIF originating from non-tumor cells might also promote pancreatic tumor growth in vivo. Less
Rational structure-based drug design SBDD relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process While SBDD has proven successful for many drug-discovery projects its application to G protein-coupled receptors GPCRs has been limited owing to extreme difficulties with their crystallization Here a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands taking advantage of the serial femtosecond crystallography approach which obviates the need for large crystals and ... More
Rational structure-based drug design (SBDD) relies on the availability of a large number of co-crystal structures to map the ligand-binding pocket of the target protein and use this information for lead-compound optimization via an iterative process. While SBDD has proven successful for many drug-discovery projects, its application to G protein-coupled receptors (GPCRs) has been limited owing to extreme difficulties with their crystallization. Here, a method is presented for the rapid determination of multiple co-crystal structures for a target GPCR in complex with various ligands, taking advantage of the serial femtosecond crystallography approach, which obviates the need for large crystals and requires only submilligram quantities of purified protein. The method was applied to the human β2-adrenergic receptor, resulting in eight room-temperature co-crystal structures with six different ligands, including previously unreported structures with carvedilol and propranolol. The generality of the proposed method was tested with three other receptors. This approach has the potential to enable SBDD for GPCRs and other difficult-to-crystallize membrane proteins. Less
Musashi- MSI belongs to Musashi family of RNA binding proteins RBP Like Musashi- MSI it is overexpressed in a variety of cancers and is a promising therapeutic target Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs Previously we have identified several inhibitors of MSI all of which bind to MSI as well In order to design MSI -specific inhibitors and compare the differences of binding mode of the inhibitors we set out to solve the structure of MSI -RRM the key motif that is responsible for the binding Here we ... More
Musashi-2 (MSI2) belongs to Musashi family of RNA binding proteins (RBP). Like Musashi-1 (MSI1), it is overexpressed in a variety of cancers and is a promising therapeutic target. Both MSI proteins contain two N-terminal RNA recognition motifs and play roles in posttranscriptional regulation of target mRNAs. Previously, we have identified several inhibitors of MSI1, all of which bind to MSI2 as well. In order to design MSI2-specific inhibitors and compare the differences of binding mode of the inhibitors, we set out to solve the structure of MSI2-RRM1, the key motif that is responsible for the binding. Here, we report the crystal structure and the first NMR solution structure of MSI2-RRM1, and compare these to the structures of MSI1-RBD1 and other RBPs. A high degree of structural similarity was observed between the crystal and solution NMR structures. MSI2-RRM1 shows a highly similar overall folding topology to MSI1-RBD1 and other RBPs. The structural information of MSI2-RRM1 will be helpful for understanding MSI2-RNA interaction and for guiding rational drug design of MSI2-specific inhibitors. Less
The G protein coupled cysteinyl leukotriene receptor CysLT R mediates inflammatory processes and plays a major role in numerous disorders including asthma allergic rhinitis cardiovascular disease and cancer Selective CysLT R antagonists are widely prescribed as antiasthmatic drugs however these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects To gain deeper understanding into the functional mechanisms of CysLTRs we determined the crystal structures of CysLT R bound to two chemically distinct antagonists zafirlukast and pranlukast The structures reveal unique ligand-binding modes and signaling mechanisms including lateral ligand access to the orthosteric pocket between transmembrane ... More
The G protein–coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT1R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue–coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs. Less
A H N virus predominated recent influenza seasons which has resulted in the rigorous investigation of haemagglutinin but whether neuraminidase NA has undergone antigenic change and contributed to the predominance of A H N virus is unknown Here we show that the NA of the circulating A H N viruses has experienced significant antigenic drift since compared with the A Hong Kong vaccine strain This antigenic drift was mainly caused by amino acid mutations at NA residues S N S T introducing an N-linked glycosylation site at residue and As a result the binding of the NA of A H ... More
A(H3N2) virus predominated recent influenza seasons, which has resulted in the rigorous investigation of haemagglutinin, but whether neuraminidase (NA) has undergone antigenic change and contributed to the predominance of A(H3N2) virus is unknown. Here, we show that the NA of the circulating A(H3N2) viruses has experienced significant antigenic drift since 2016 compared with the A/Hong Kong/4801/2014 vaccine strain. This antigenic drift was mainly caused by amino acid mutations at NA residues 245, 247 (S245N/S247T; introducing an N-linked glycosylation site at residue 245) and 468. As a result, the binding of the NA of A(H3N2) virus by some human monoclonal antibodies, including those that have broad reactivity to the NA of the 1957 A(H2N2) and 1968 A(H3N2) reference pandemic viruses as well as contemporary A(H3N2) strains, was reduced or abolished. This antigenic drift also reduced NA-antibody-based protection against in vivo virus challenge. X-ray crystallography showed that the glycosylation site at residue 245 is within a conserved epitope that overlaps the NA active site, explaining why it impacts antibody binding. Our findings suggest that NA antigenic drift impacts protection against influenza virus infection, thus highlighting the importance of including NA antigenicity for consideration in the optimization of influenza vaccines. Less
Human anti-HIV- broadly neutralizing antibodies bNAbs protect against infection in animal models However bNAbs have not been elicited by vaccination in diverse wild-type animals or humans in part because B cells expressing the precursors of these antibodies do not recognize most HIV- envelopes Envs Immunogens have been designed that activate these B cell precursors in vivo but they also activate competing off-target responses Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody iv that selects for naive human B cells expressing immunoglobulin light chains with amino acid complementarity determining region s a key ... More
Human anti-HIV-1 broadly neutralizing antibodies (bNAbs) protect against infection in animal models. However, bNAbs have not been elicited by vaccination in diverse wild-type animals or humans, in part because B cells expressing the precursors of these antibodies do not recognize most HIV-1 envelopes (Envs). Immunogens have been designed that activate these B cell precursors in vivo, but they also activate competing off-target responses. Here we report on a complementary approach to expand specific B cells using an anti-idiotypic antibody, iv8, that selects for naive human B cells expressing immunoglobulin light chains with 5–amino acid complementarity determining region 3s, a key feature of anti-CD4 binding site (CD4bs)–specific VRC01-class antibodies. In mice, iv8 induced target cells to expand and mature in the context of a polyclonal immune system and produced serologic responses targeting the CD4bs on Env. In summary, the results demonstrate that an anti-idiotypic antibody can specifically recognize and expand rare B cells that express VRC01-class antibodies against HIV-1. Less
Detection and activation of HIV broadly neutralizing antibody precursor B cells using anti-idiotypes
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest In light of this there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes Here we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus HIV- broadly neutralizing antibody b iglb We determined the crystal structure of two anti-idiotypes in complex with iglb and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb Immunization ... More
Many tested vaccines fail to provide protection against disease despite the induction of antibodies that bind the pathogen of interest. In light of this, there is much interest in rationally designed subunit vaccines that direct the antibody response to protective epitopes. Here, we produced a panel of anti-idiotype antibodies able to specifically recognize the inferred germline version of the human immunodeficiency virus 1 (HIV-1) broadly neutralizing antibody b12 (iglb12). We determined the crystal structure of two anti-idiotypes in complex with iglb12 and used these anti-idiotypes to identify rare naive human B cells expressing B cell receptors with similarity to iglb12. Immunization with a multimerized version of this anti-idiotype induced the proliferation of transgenic murine B cells expressing the iglb12 heavy chain in vivo, despite the presence of deletion and anergy within this population. Together, our data indicate that anti-idiotypes are a valuable tool for the study and induction of potentially protective antibodies. Less
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another Here histidine residues are introduced into the hydrophobic core of a coiled-coil trimer generating a peptide that self-assembles upon the addition of metal ions HisAD the resulting peptide is unstructured in the absence of metal and folds selectively to form an -helical construct upon complexation with Cu II and Ni II but not Co II or Zn II The structure and metal-binding ability of HisAD is probed using a combination of circular dichroism CD spectroscopy analytical ... More
Designing peptides that fold and assemble in response to metal ions tests our understanding of how peptide folding and metal binding influence one another. Here, histidine residues are introduced into the hydrophobic core of a coiled-coil trimer, generating a peptide that self-assembles upon the addition of metal ions. HisAD, the resulting peptide, is unstructured in the absence of metal and folds selectively to form an α-helical construct upon complexation with Cu(II) and Ni(II) but not Co(II) or Zn(II). The structure, and metal-binding ability, of HisAD is probed using a combination of circular dichroism (CD) spectroscopy, analytical ultracentrifugation (AUC), nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These show the peptide is trimeric and binds to both Cu(II) and Ni(II) in a 1 : 1 ratio with the histidine residues involved in the metal coordination, as designed. The X-ray crystal structure of the HisAD-Cu(II) complex reveals the trimeric HisAD peptide coordinates three Cu(II) ions; this is the first example of such a structure. Additionally, HisAD demonstrates an unprecedented discrimination between transition metal ions, the basis of which is likely to be related to the stability of the peptide-metal complexes formed. Less
Toxin-antitoxin TA gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance TA loci are classified into various types based upon the characteristics of their antitoxins with those in type II expressing proteic antitoxins Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal PIN domain containing conserved amino acids considered essential for activity The vapBC virulence-associated protein TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae NTHi In this study the crystal structure of ... More
Toxin-antitoxin (TA) gene pairs have been identified in nearly all bacterial genomes sequenced to date and are thought to facilitate persistence and antibiotic tolerance. TA loci are classified into various types based upon the characteristics of their antitoxins, with those in type II expressing proteic antitoxins. Many toxins from type II modules are ribonucleases that maintain a PilT N-terminal (PIN) domain containing conserved amino acids considered essential for activity. The vapBC (virulence-associated protein) TA system is the largest subfamily in this class and has been linked to pathogenesis of nontypeable Haemophilus influenzae (NTHi). In this study, the crystal structure of the VapBC-1 complex from NTHi was determined to 2.20 Å resolution. Based on this structure, aspartate-to-asparagine and glutamate-to-glutamine mutations of four conserved residues in the PIN domain of the VapC-1 toxin were constructed and the effects of the mutations on protein-protein interactions, growth of Escherichia coli, and pathogenesis ex vivo were tested. Finally, a novel model system was designed and utilized that consists of an NTHi ΔvapBC-1 strain complemented in cis with the TA module containing a mutated or wild-type toxin at an ectopic site on the chromosome. This enabled the analysis of the effect of PIN domain toxin mutants in tandem with their wild-type antitoxin under the control of the vapBC-1 native promoter and in single copy. This is the first report of a system facilitating the study of TA mutant operons in the background of NTHi during infections of primary human tissues ex vivo. Less
Melatonin N-acetyl- -methoxytryptamine is a neurohormone that maintains circadian rhythms by synchronization to environmental cues and is involved in diverse physiological processes such as the regulation of blood pressure and core body temperature oncogenesis and immune function Melatonin is formed in the pineal gland in a light-regulated manner by enzymatic conversion from -hydroxytryptamine -HT or serotonin and modulates sleep and wakefulness by activating two high-affinity G-protein-coupled receptors type A MT and type B MT Shift work travel and ubiquitous artificial lighting can disrupt natural circadian rhythms as a result sleep disorders affect a substantial population in modern society and pose ... More
Melatonin (N-acetyl-5-methoxytryptamine) is a neurohormone that maintains circadian rhythms1 by synchronization to environmental cues and is involved in diverse physiological processes2 such as the regulation of blood pressure and core body temperature, oncogenesis, and immune function3. Melatonin is formed in the pineal gland in a light-regulated manner4 by enzymatic conversion from 5-hydroxytryptamine (5-HT or serotonin), and modulates sleep and wakefulness5 by activating two high-affinity G-protein-coupled receptors, type 1A (MT1) and type 1B (MT2)3,6. Shift work, travel, and ubiquitous artificial lighting can disrupt natural circadian rhythms; as a result, sleep disorders affect a substantial population in modern society and pose a considerable economic burden7. Over-the-counter melatonin is widely used to alleviate jet lag and as a safer alternative to benzodiazepines and other sleeping aids8,9, and is one of the most popular supplements in the United States10. Here, we present high-resolution room-temperature X-ray free electron laser (XFEL) structures of MT1 in complex with four agonists: the insomnia drug ramelteon11, two melatonin analogues, and the mixed melatonin–serotonin antidepressant agomelatine12,13. The structure of MT2 is described in an accompanying paper14. Although the MT1 and 5-HT receptors have similar endogenous ligands, and agomelatine acts on both receptors, the receptors differ markedly in the structure and composition of their ligand pockets; in MT1, access to the ligand pocket is tightly sealed from solvent by extracellular loop 2, leaving only a narrow channel between transmembrane helices IV and V that connects it to the lipid bilayer. The binding site is extremely compact, and ligands interact with MT1 mainly by strong aromatic stacking with Phe179 and auxiliary hydrogen bonds with Asn162 and Gln181. Our structures provide an unexpected example of atypical ligand entry for a non-lipid receptor, lay the molecular foundation of ligand recognition by melatonin receptors, and will facilitate the design of future tool compounds and therapeutic agents, while their comparison to 5-HT receptors yields insights into the evolution and polypharmacology of G-protein-coupled receptors. Less
The human MT and MT melatonin receptors are G protein-coupled receptors GPCRs involved in the regulation of circadian rhythm and sleep patterns Drug development efforts target both receptors for treatment of insomnia circadian rhythm and mood disorders and cancer while MT has also been implicated in type diabetes T D Here we report the X-ray Free Electron Laser XFEL structures of the human MT receptor in complex with agonists -phenylmelatonin -pmt and ramelteon at resolutions of and respectively along with two structures of function-related mutants H A superscripts represent the Ballesteros-Weinstein residue numbering nomenclature and N D obtained in complex ... More
The human MT11 and MT22 melatonin receptors are G protein-coupled receptors (GPCRs) involved in the regulation of circadian rhythm and sleep patterns3. Drug development efforts target both receptors for treatment of insomnia, circadian rhythm and mood disorders, and cancer3, while MT2 has also been implicated in type 2 diabetes (T2D)4,5. Here we report the X-ray Free Electron Laser (XFEL) structures of the human MT2 receptor in complex with agonists 2-phenylmelatonin (2-pmt) and ramelteon6 at resolutions of 2.8 Å and 3.3 Å, respectively, along with two structures of function-related mutants, H2085.46A (superscripts represent the Ballesteros-Weinstein residue numbering nomenclature7) and N862.50D, obtained in complex with 2-pmt. Comparison of the MT2 structures with MT18 reveals that, despite the fact that the orthosteric ligand-binding site residues are conserved, there are notable conformational variations as well as differences in [3H]-melatonin dissociation kinetics that provide new insights into the selectivity between melatonin receptor subtypes. In addition to the membrane-buried lateral ligand entry channel that is also observed in MT1, the MT2 structures reveal a narrow opening towards the solvent in the extracellular part of the receptor. We provide functional and kinetic data supporting a prominent role for the intramembrane ligand entry in both receptors, while simultaneously suggesting the possibility of an extracellular entry path in MT2. Our findings contribute to a molecular understanding of melatonin receptor subtype selectivity and ligand access modes, which are essential for the design of highly selective melatonin tool compounds and therapeutic agents. Less
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications Recently the first sodium-pumping rhodopsin KR from Krokinobacter eikastus was discovered and characterized However the existing structures of KR are contradictory and the mechanism of Na pumping is not yet understood Here we present a structure of the cationic non H light-driven pump at physiological pH in its pentameric form We also present atomic structures and functional data on the KR and its mutants including potassium pumps which show that oligomerization of the microbial ... More
Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics. Less
The hyperthermophilic crenarchaeon Ignicoccus hospitalis KIN I possesses at least putative genes encoding enzymes that belong to the -hydrolase superfamily One of those genes the metallo-hydrolase-encoding igni was cloned and heterologously expressed in Pichia pastoris The enzyme produced was purified in its catalytically active form The recombinant enzyme was successfully crystallized and the crystal diffracted to a resolution of The crystal belonged to space group R with unit-cell parameters a b c It is suggested that it contains one monomer of Igni within the asymmetric unit
NEMO is an essential component in the activation of the canonical NF- B pathway and exerts its function by recruiting the I B kinases IKK to the IKK complex Inhibition of the NEMO IKKs interaction is an attractive therapeutic paradigm for diseases related to NF- B mis-regulation but a difficult endeavor because of the extensive protein-protein interface Here we report the high-resolution structure of the unbound IKK -binding domain of NEMO that will greatly facilitate the design of NEMO IKK inhibitors The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a ... More
NEMO is an essential component in the activation of the canonical NF-?B pathway and exerts its function by recruiting the I?B kinases (IKK) to the IKK complex. Inhibition of the NEMO/IKKs interaction is an attractive therapeutic paradigm for diseases related to NF-?B mis-regulation, but a difficult endeavor because of the extensive protein-protein interface. Here we report the high-resolution structure of the unbound IKK�-binding domain of NEMO that will greatly facilitate the design of NEMO/IKK inhibitors. The structures of unbound NEMO show a closed conformation that partially occludes the three binding hot-spots and suggest a facile transition to an open state that can accommodate ligand binding. By fusing coiled-coil adaptors to the IKK�-binding domain of NEMO, we succeeded in creating a protein with improved solution behavior, IKK�-binding affinity and crystallization compatibility, which will enable the structural characterization of new NEMO/inhibitor complexes. Less
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors GPCRs Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A A adenosine receptor A AAR D N and S A Both structures present an overall active-like conformation however the variants show key changes in the activation motif NPxxY Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability ... More
Sodium ions are endogenous allosteric modulators of many G protein-coupled receptors (GPCRs). Mutation of key residues in the sodium binding motif causes a striking effect on G protein signaling. We report the crystal structures of agonist complexes for two variants in the first sodium coordination shell of the human A2A adenosine receptor (A2AAR), D522.50N and S913.39A. Both structures present an overall active-like conformation; however, the variants show key changes in the activation motif NPxxY. Changes in the hydrogen bonding network in this microswitch suggest a possible mechanism for modified G protein signaling and enhanced thermal stability. These structures, signaling data, and thermal stability analysis with a panel of pharmacological ligands provide a basis for understanding the role of the sodium-coordinating residues on stability and G protein signaling. Utilizing the D2.50N variant is a promising method for stabilizing class A GPCRs to accelerate structural efforts and drug discovery. Less
Nramp family transporters expressed in organisms from bacteria to humans enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport We present high-resolution structures of Deinococcus radiodurans Dra Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere Strikingly while metal transport requires cycling from outward- to inward-open states efficient proton transport still occurs in outward-locked but not inward-locked DraNramp We propose a model in which metal and proton enter the transporter via the same external pathway ... More
Nramp family transporters—expressed in organisms from bacteria to humans—enable uptake of essential divalent transition metals via an alternating-access mechanism that also involves proton transport. We present high-resolution structures of Deinococcus radiodurans (Dra)Nramp in multiple conformations to provide a thorough description of the Nramp transport cycle by identifying the key intramolecular rearrangements and changes to the metal coordination sphere. Strikingly, while metal transport requires cycling from outward- to inward-open states, efficient proton transport still occurs in outward-locked (but not inward-locked) DraNramp. We propose a model in which metal and proton enter the transporter via the same external pathway to the binding site, but follow separate routes to the cytoplasm, which could facilitate the co-transport of two cationic species. Our results illustrate the flexibility of the LeuT fold to support a broad range of substrate transport and conformational change mechanisms. Less
Many drugs target the serotonin A receptor -HT AR including second-generation antipsychotics that also target the dopamine D receptor D R These drugs often produce severe side effects due to non-selective binding to other aminergic receptors Here we report the structures of human -HT AR in complex with the second-generation antipsychotics risperidone and zotepine These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket the movements of which are important for receptor activation -HT AR is structurally similar to -HT CR but possesses a unique side-extended cavity near the ... More
Many drugs target the serotonin 2A receptor (5-HT2AR), including second-generation antipsychotics that also target the dopamine D2 receptor (D2R). These drugs often produce severe side effects due to non-selective binding to other aminergic receptors. Here, we report the structures of human 5-HT2AR in complex with the second-generation antipsychotics risperidone and zotepine. These antipsychotics effectively stabilize the inactive conformation by forming direct contacts with the residues at the bottom of the ligand-binding pocket, the movements of which are important for receptor activation. 5-HT2AR is structurally similar to 5-HT2CR but possesses a unique side-extended cavity near the orthosteric binding site. A docking study and mutagenic studies suggest that a highly 5-HT2AR-selective antagonist binds the side-extended cavity. The conformation of the ligand-binding pocket in 5-HT2AR significantly differs around extracellular loops 1 and 2 from that in D2R. These findings are beneficial for the rational design of safer antipsychotics and 5-HT2AR-selective drugs. Less
Mycobacterium tuberculosis Mtb the main causative agent of tuberculosis TB is naturally resistant to -lactam antibiotics due to the production of the extended spectrum -lactamase BlaC -Lactam -lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB However still little is known of the exact mechanism of BlaC inhibition by the -lactamase inhibitors currently approved for clinical use clavulanic acid sulbactam tazobactam and avibactam Here we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors The Da adduct derived from clavulanate and the trans-enamine ... More
Mycobacterium tuberculosis (Mtb), the main causative agent of tuberculosis (TB), is naturally resistant to β-lactam antibiotics due to the production of the extended spectrum β-lactamase BlaC. β-Lactam/β-lactamase inhibitor combination therapies can circumvent the BlaC-mediated resistance of Mtb and are promising treatment options against TB. However, still little is known of the exact mechanism of BlaC inhibition by the β-lactamase inhibitors currently approved for clinical use, clavulanic acid, sulbactam, tazobactam, and avibactam. Here, we present the X-ray diffraction crystal structures of the acyl-enzyme adducts of wild-type BlaC with the four inhibitors. The +70 Da adduct derived from clavulanate and the trans-enamine acylation adducts of sulbactam and tazobactam are reported. BlaC in complex with avibactam revealed two inhibitor conformations. Preacylation binding could not be observed because inhibitor binding was not detected in BlaC variants carrying a substitution of the active site serine 70 to either alanine or cysteine, by crystallography, ITC or NMR. These results suggest that the catalytic serine 70 is necessary not only for enzyme acylation but also for increasing BlaC affinity for inhibitors in the preacylation state. The structure of BlaC with the serine to cysteine mutation showed a covalent linkage of the cysteine 70 Sγ atom to the nearby amino group of lysine 73. The differences of adduct conformations between BlaC and other β-lactamases are discussed. Less
Prostaglandin E receptor EP a G-protein-coupled receptor is involved in disorders such as cancer and autoimmune disease Here we report the crystal structure of human EP in complex with its antagonist ONO-AE - and an inhibitory antibody at resolution The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer proximal to the highly conserved Arg residue in the seventh transmembrane domain Functional and docking studies demonstrate that the natural agonist PGE binds in a similar manner This structural information also provides insight into the ligand ... More
Prostaglandin E receptor EP4, a G-protein-coupled receptor, is involved in disorders such as cancer and autoimmune disease. Here, we report the crystal structure of human EP4 in complex with its antagonist ONO-AE3-208 and an inhibitory antibody at 3.2 Å resolution. The structure reveals that the extracellular surface is occluded by the extracellular loops and that the antagonist lies at the interface with the lipid bilayer, proximal to the highly conserved Arg316 residue in the seventh transmembrane domain. Functional and docking studies demonstrate that the natural agonist PGE2 binds in a similar manner. This structural information also provides insight into the ligand entry pathway from the membrane bilayer to the EP4 binding pocket. Furthermore, the structure reveals that the antibody allosterically affects the ligand binding of EP4. These results should facilitate the design of new therapeutic drugs targeting both orthosteric and allosteric sites in this receptor family. Less
Toxin antitoxin TA systems are involved in diverse physiological processes in prokaryotes but their exact role in Mycobacterium tuberculosis Mtb virulence and in vivo stress adaptation has not been extensively studied Here we demonstrate that the VapBC TA module is essential for Mtb to establish infection in guinea pigs RNA-sequencing revealed that overexpression of VapC toxin results in metabolic slowdown suggesting that modulation of the growth rate is an essential strategy for in vivo survival Interestingly overexpression of VapC resulted in the upregulation of chromosomal TA genes suggesting the existence of highly coordinated crosstalk among TA systems In this study ... More
Toxin–antitoxin (TA) systems are involved in diverse physiological processes in prokaryotes, but their exact role in Mycobacterium tuberculosis (Mtb) virulence and in vivo stress adaptation has not been extensively studied. Here, we demonstrate that the VapBC11 TA module is essential for Mtb to establish infection in guinea pigs. RNA-sequencing revealed that overexpression of VapC11 toxin results in metabolic slowdown, suggesting that modulation of the growth rate is an essential strategy for in vivo survival. Interestingly, overexpression of VapC11 resulted in the upregulation of chromosomal TA genes, suggesting the existence of highly coordinated crosstalk among TA systems. In this study, we also present the crystal structure of the VapBC11 heterooctameric complex at 1.67 Å resolution. Binding kinetic studies suggest that the binding affinities of toxin–substrate and toxin–antitoxin interactions are comparable. We used a combination of structural studies, molecular docking, mutational analysis and in vitro ribonuclease assays to enhance our understanding of the mode of substrate recognition by the VapC11 toxin. Furthermore, we have also designed peptide-based inhibitors to target VapC11 ribonuclease activity. Taken together, we propose that the structure-guided design of inhibitors against in vivo essential ribonucleases might be a novel strategy to hasten clearance of intracellular Mtb. Less
Human respiratory syncytial virus HRSV is a negative-stranded RNA virus that causes a globally prevalent respiratory infection which can cause lifethreatening illness particularly in the young elderly and immunocompromised HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase RdRp For replication this complex comprises the phosphoprotein P and the large protein L whereas for transcription the M - protein is also required M - is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M - surface such that binding of these ... More
Human respiratory syncytial virus (HRSV) is a negative-stranded RNA virus that causes a globally prevalent respiratory infection, which can cause lifethreatening illness, particularly in the young, elderly, and immunocompromised. HRSV multiplication depends on replication and transcription of the HRSV genes by the virus-encoded RNA-dependent RNA polymerase (RdRp). For replication, this complex comprises the phosphoprotein (P) and the large protein (L), whereas for transcription, the M2-1 protein is also required. M2-1 is recruited to the RdRp by interaction with P and also interacts with RNA at overlapping binding sites on the M2-1 surface, such that binding of these partners is mutually exclusive. The molecular basis for the transcriptional requirement of M2-1 is unclear, as is the consequence of competition between P and RNA for M2-1 binding, which is likely a critical step in the transcription mechanism. Here, we report the crystal structure at 2.4 Å of M2-1 bound to the P interaction domain, which comprises P residues 90 to 110. The P90 – 110 peptide is alpha helical, and its position on the surface of M2-1 defines the orientation of the three transcriptase components within the complex. The M2-1/P interface includes ionic, hydrophobic, and hydrogen bond interactions, and the critical contribution of these contacts to complex formation was assessed using a minigenome assay. The affinity of M2-1 for RNA and P ligands was quantified using fluorescence anisotropy, which showed high-affinity RNAs could outcompete P. This has important implications for the mechanism of transcription, particularly the events surrounding transcription termination and synthesis of poly(A) sequences. Less
Human muscarinic receptor M is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors Muscarinic receptors are targets for multiple neurodegenerative diseases The challenge has been designing subtype selective ligands against one of the five muscarinic receptors We report high resolution structures of a thermostabilized mutant M receptor bound to a subtype selective antagonist AF-DX and a non-selective antagonist NMS The thermostabilizing mutation S R in M was predicted using a theoretical strategy previously developed in our group Comparison of the crystal structures and pharmacological properties of the M receptor shows that the ... More
Human muscarinic receptor, M2 is one of the five subtypes of muscarinic receptors belonging to the family of G protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype selective ligands against one of the five muscarinic receptors. We report high resolution structures of a thermostabilized mutant M2 receptor bound to a subtype selective antagonist AF-DX 384 and a non-selective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, that is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular Dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptor compared to M3 receptor leads to subtype selectivity of AF-DX 384. Less
Kinesin- transports numerous cellular cargoes along microtubules The kinesin- light chain KLC mediates cargo binding and regulates kinesin- motility To investigate the molecular basis for kinesin- recruitment and activation by cargoes we solved the crystal structure of the KLC tetratricopeptide repeat TPR domain bound to the cargo JIP This combined with biophysical and molecular evolutionary analyses reveals a kinesin- cargo binding site located on KLC TPR which is conserved in homologs from sponges to humans In the complex JIP crosslinks two KLC TPR domains via their TPR s We show that TPR forms a dimer interface that mimics JIP binding ... More
Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. Less
Frizzled receptors FZDs are class-F G-protein-coupled receptors GPCRs that function in Wnt signalling and are essential for developing and adult organisms As central mediators in this complex signalling pathway FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research Here we present an atomic-resolution structure of the human Frizzled receptor FZD transmembrane domain in the absence of a bound ligand The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed and is distinct from all other GPCR structures reported so far Within this ... More
Frizzled receptors (FZDs) are class-F G-protein-coupled receptors (GPCRs) that function in Wnt signalling and are essential for developing and adult organisms1,2. As central mediators in this complex signalling pathway, FZDs serve as gatekeeping proteins both for drug intervention and for the development of probes in basic and in therapeutic research. Here we present an atomic-resolution structure of the human Frizzled 4 receptor (FZD4) transmembrane domain in the absence of a bound ligand. The structure reveals an unusual transmembrane architecture in which helix VI is short and tightly packed, and is distinct from all other GPCR structures reported so far. Within this unique transmembrane fold is an extremely narrow and highly hydrophilic pocket that is not amenable to the binding of traditional GPCR ligands. We show that such a pocket is conserved across all FZDs, which may explain the long-standing difficulties in the development of ligands for these receptors. Molecular dynamics simulations on the microsecond timescale and mutational analysis uncovered two coupled, dynamic kinks located at helix VII that are involved in FZD4 activation. The stability of the structure in its ligand-free form, an unfavourable pocket for ligand binding and the two unusual kinks on helix VII suggest that FZDs may have evolved a novel ligand-recognition and activation mechanism that is distinct from that of other GPCRs. Less
The avian influenza A H N virus continues to cause human infections in China and is a major ongoing public health concern Five epidemic waves of A H N infection have occurred since and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves Moreover highly pathogenic avian influenza HPAI A H N viruses were also isolated during the fifth epidemic wave Here we present a detailed structural and biochemical analysis of the surface hemagglutinin HA antigen ... More
The avian influenza A(H7N9) virus continues to cause human infections in China and is a major ongoing public health concern. Five epidemic waves of A(H7N9) infection have occurred since 2013, and the recent fifth epidemic wave saw the emergence of two distinct lineages with elevated numbers of human infection cases and broader geographic distribution of viral diseases compared to the first four epidemic waves. Moreover, highly pathogenic avian influenza (HPAI) A(H7N9) viruses were also isolated during the fifth epidemic wave. Here, we present a detailed structural and biochemical analysis of the surface hemagglutinin (HA) antigen from viruses isolated during this recent epidemic wave. Results highlight that, compared to the 2013 virus HAs, the fifth-wave virus HAs remained a weak binder to human glycan receptor analogs. We also studied three mutations, V177K-K184T-G219S, that were recently reported to switch a 2013 A(H7N9) HA to human-type receptor specificity. Our results indicate that these mutations could also switch the H7 HA receptor preference to a predominantly human binding specificity for both fifth-wave H7 HAs analyzed in this study. Less
Angiotensin II AngII plays a central role in regulating human blood pressure which is mainly mediated by interactions between AngII and the G-protein-coupled receptors GPCRs AngII type receptor AT R and AngII type receptor AT R We have solved the crystal structure of human AT R binding the peptide ligand Sar Ile AngII and its specific antibody at - resolution Sar Ile AngII interacts with both the core binding domain where the small-molecule ligands of AT R and AT R bind and the extended binding domain which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor We ... More
Angiotensin II (AngII) plays a central role in regulating human blood pressure, which is mainly mediated by interactions between AngII and the G-protein-coupled receptors (GPCRs) AngII type 1 receptor (AT1R) and AngII type 2 receptor (AT2R). We have solved the crystal structure of human AT2R binding the peptide ligand [Sar1, Ile8]AngII and its specific antibody at 3.2-Å resolution. [Sar1, Ile8]AngII interacts with both the ‘core’ binding domain, where the small-molecule ligands of AT1R and AT2R bind, and the ‘extended’ binding domain, which is equivalent to the allosteric modulator binding site of muscarinic acetylcholine receptor. We generated an antibody fragment to stabilize the extended binding domain that functions as a positive allosteric modulator. We also identified a signature positively charged cluster, which is conserved among peptide-binding receptors, to locate C termini at the bottom of the binding pocket. The reported results should help with designing ligands for angiotensin receptors and possibly to other peptide GPCRs. Less
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality However we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity which is critically required to enable rational design of degraders Here we utilize comprehensive characterization of the ligand dependent CRBN BRD interaction to demonstrate that binding between proteins that have not evolved to interact is plastic Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations which are selectively bound by ligands We demonstrate that computational protein-protein docking can ... More
Heterobifunctional small molecule degraders that induce protein degradation through ligase-mediated ubiquitination have shown considerable promise as a new pharmacological modality. However, we currently lack a detailed understanding of the molecular basis for target recruitment and selectivity, which is critically required to enable rational design of degraders. Here we utilize comprehensive characterization of the ligand dependent CRBN/BRD4 interaction to demonstrate that binding between proteins that have not evolved to interact is plastic. Multiple X-ray crystal structures show that plasticity results in several distinct low energy binding conformations, which are selectively bound by ligands. We demonstrate that computational protein-protein docking can reveal the underlying inter-protein contacts and inform the design of a BRD4 selective degrader that can discriminate between highly homologous BET bromodomains. Our findings that plastic inter-protein contacts confer selectivity for ligand-induced protein dimerization provide a conceptual framework for the development of heterobifunctional ligands. Less
-Galactosidases EC are retaining glycosidases that cleave terminal -linked galactose residues from glycoconjugate substrates -Galactosidases take part in the turnover of cell wall associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals Deficiency of human -galactosidase A -Gal A causes Fabry disease FD a heritable X-linked lysosomal storage disorder characterized by accumulation of globotriaosylceramide Gb and globotriaosylsphingosine lyso-Gb Current management of FD involves enzyme-replacement therapy ERT An activity-based probe ABP covalently labeling the catalytic nucleophile of -Gal A has been previously designed to study -galactosidases for use in FD therapy Here we report that this ABP ... More
α-Galactosidases (EC 3.2.1.22) are retaining glycosidases that cleave terminal α-linked galactose residues from glycoconjugate substrates. α-Galactosidases take part in the turnover of cell wall–associated galactomannans in plants and in the lysosomal degradation of glycosphingolipids in animals. Deficiency of human α-galactosidase A (α-Gal A) causes Fabry disease (FD), a heritable, X-linked lysosomal storage disorder, characterized by accumulation of globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). Current management of FD involves enzyme-replacement therapy (ERT). An activity-based probe (ABP) covalently labeling the catalytic nucleophile of α-Gal A has been previously designed to study α-galactosidases for use in FD therapy. Here, we report that this ABP labels proteins in Nicotiana benthamiana leaf extracts, enabling the identification and biochemical characterization of an N. benthamiana α-galactosidase we name here A1.1 (gene accession ID GJZM-1660). The transiently overexpressed and purified enzyme was a monomer lacking N-glycans and was active toward 4-methylumbelliferyl-α-d-galactopyranoside substrate (Km = 0.17 mm) over a broad pH range. A1.1 structural analysis by X-ray crystallography revealed marked similarities with human α-Gal A, even including A1.1's ability to hydrolyze Gb3 and lyso-Gb3, which are not endogenous in plants. Of note, A1.1 uptake into FD fibroblasts reduced the elevated lyso-Gb3 levels in these cells, consistent with A1.1 delivery to lysosomes as revealed by confocal microscopy. The ease of production and the features of A1.1, such as stability over a broad pH range, combined with its capacity to degrade glycosphingolipid substrates, warrant further examination of its value as a potential therapeutic agent for ERT-based FD management. Less
Epstein-Barr virus EBV is a causative agent of infectious mononucleosis and is associated with new cases of cancer and deaths annually Subunit vaccines against this pathogen have focused on the gp glycoprotein and remain unsuccessful We isolated human antibodies recognizing the EBV fusion machinery gH gL and gB from rare memory B cells One anti-gH gL antibody AMMO potently neutralized infection of B cells and epithelial cells the two major cell types targeted by EBV We determined a cryo-electron microscopy reconstruction of the gH gL-gp -AMMO complex and demonstrated that AMMO bound to a discontinuous epitope formed by both gH ... More
Epstein-Barr virus (EBV) is a causative agent of infectious mononucleosis and is associated with 200,000 new cases of cancer and 140,000 deaths annually. Subunit vaccines against this pathogen have focused on the gp350 glycoprotein and remain unsuccessful. We isolated human antibodies recognizing the EBV fusion machinery (gH/gL and gB) from rare memory B cells. One anti-gH/gL antibody, AMMO1, potently neutralized infection of B cells and epithelial cells; the two major cell types targeted by EBV. We determined a cryo-electron microscopy reconstruction of the gH/gL-gp42-AMMO1 complex and demonstrated that AMMO1 bound to a discontinuous epitope formed by both gH and gL at the Domain-I/Domain-II interface. Integrating structural, biochemical and infectivity data, we propose that AMMO1 inhibits fusion of the viral and cellular membranes. This work identifies a crucial epitope that may aid in the design of next-generation subunit vaccines against this major public health burden. Less
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein related lipid transfer StART domains that are implicated in intracellular non-vesicular lipid transport A new family of membrane-anchored StARkins was recently identified including six members Lam Lam in the yeast Saccharomyces cerevisiae Lam Lam are anchored to the endoplasmic reticulum ER membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast To better understand the biological roles of these proteins we carried out a structure-function analysis of the second StARkin domain of Lam here termed Lam S NMR ... More
The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein–related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1–Lam6, in the yeast Saccharomyces cerevisiae. Lam1–Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling “head-first” entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress. Less
Mitochondrial Carriers MCs are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism The ADP ATP Carriers AACs are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP In human four different AACs isoforms are described which are expressed in tissue-specific manner They are involved in different genetic diseases and play a role in cancerogenesis Up to now only the structures of the bovine isoform and yeast isoforms and AAC have been determined in one particular conformation obtained in complex ... More
Mitochondrial Carriers (MCs) are responsible for fluent traffic of a variety of compounds that need to be shuttled via mitochondrial inner membranes to maintain cell metabolism. The ADP/ATP Carriers (AACs) are responsible for the import of ADP inside the mitochondria and the export of newly synthesized ATP. In human, four different AACs isoforms are described which are expressed in tissue-specific manner. They are involved in different genetic diseases and play a role in cancerogenesis. Up to now only the structures of the bovine (isoform 1) and yeast (isoforms 2 and 3) AAC have been determined in one particular conformation, obtained in complex with the CATR inhibitor. Herein, we report that full-length human ADP/ATP Carriers isoform 1 and 3 were successfully expressed in cell-free system and purified in milligram amounts in detergent-solubilized state. The proteins exhibited the expected secondary structure content. Thermostability profiles showing stabilization by the CATR inhibitor suggest that the carriers are well folded. Less
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum Pf circumsporozoite protein PfCSP from several subjects immunized with an attenuated Pf whole-sporozoite SPZ vaccine Sanaria PfSPZ Vaccine Passive transfer of one of these antibodies monoclonal antibody CIS conferred high-level sterile protection in two different mouse models of malaria infection The affinity and stoichiometry of CIS binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain The first ... More
Development of a highly effective vaccine or antibodies for the prevention and ultimately elimination of malaria is urgently needed. Here we report the isolation of a number of human monoclonal antibodies directed against the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP) from several subjects immunized with an attenuated Pf whole-sporozoite (SPZ) vaccine (Sanaria PfSPZ Vaccine). Passive transfer of one of these antibodies, monoclonal antibody CIS43, conferred high-level, sterile protection in two different mouse models of malaria infection. The affinity and stoichiometry of CIS43 binding to PfCSP indicate that there are two sequential multivalent binding events encompassing the repeat domain. The first binding event is to a unique 'junctional' epitope positioned between the N terminus and the central repeat domain of PfCSP. Moreover, CIS43 prevented proteolytic cleavage of PfCSP on PfSPZ. Analysis of crystal structures of the CIS43 antigen-binding fragment in complex with the junctional epitope determined the molecular interactions of binding, revealed the epitope's conformational flexibility and defined Asn-Pro-Asn (NPN) as the structural repeat motif. The demonstration that CIS43 is highly effective for passive prevention of malaria has potential application for use in travelers, military personnel and elimination campaigns and identifies a new and conserved site of vulnerability on PfCSP for next-generation rational vaccine design. Less
Drugs frequently require interactions with multiple targets via a process known as polypharmacology to achieve their therapeutic actions Currently drugs targeting several serotonin receptors including the -HT C receptor are useful for treating obesity drug abuse and schizophrenia The competing challenges of developing selective -HT C receptor ligands or creating drugs with a defined polypharmacological profile especially aimed at G protein-coupled receptors GPCRs remain extremely difficult Here we solved two structures of the -HT C receptor in complex with the highly promiscuous agonist ergotamine and the -HT A-C receptor-selective inverse agonist ritanserin at resolutions of and respectively We analyzed their ... More
Drugs frequently require interactions with multiple targets — via a process known as polypharmacology — to achieve their therapeutic actions. Currently, drugs targeting several serotonin receptors, including the 5-HT2C receptor, are useful for treating obesity, drug abuse, and schizophrenia. The competing challenges of developing selective 5-HT2C receptor ligands or creating drugs with a defined polypharmacological profile, especially aimed at G protein-coupled receptors (GPCRs), remain extremely difficult. Here we solved two structures of the 5-HT2C receptor in complex with the highly promiscuous agonist ergotamine and the 5-HT2A-C receptor-selective inverse agonist ritanserin, at resolutions of 3.0 Å and 2.7 Å, respectively. We analyzed their respective binding poses to provide mechanistic insights into their receptor recognition and opposing pharmacological actions. This study investigates the structural basis of polypharmacology at canonical GPCRs and illustrates how understanding characteristic patterns of ligand-receptor interaction and activation may ultimately facilitate drug design at multiple GPCRs. Less
Protein crystallography has significantly advanced in recent years with in situ data collection in which crystals are placed in the X-ray beam within their growth medium being a major point of focus In situ methods eliminate the need to harvest crystals a previously unavoidable drawback particularly for often small membrane-protein crystals Here we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature C and under cryogenic conditions The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive easy to make and compatible with automated ... More
Protein crystallography has significantly advanced in recent years, with in situ data collection, in which crystals are placed in the X-ray beam within their growth medium, being a major point of focus. In situ methods eliminate the need to harvest crystals, a previously unavoidable drawback, particularly for often small membrane-protein crystals. Here, we present a protocol for the high-throughput in situ X-ray screening of and data collection from soluble and membrane-protein crystals at room temperature (20–25°C) and under cryogenic conditions. The Mylar in situ method uses Mylar-based film sandwich plates that are inexpensive, easy to make, and compatible with automated imaging, and that show very low background scattering. They support crystallization in microbatch and vapor-diffusion modes, as well as in lipidic cubic phases (LCPs). A set of 3D-printed holders for differently sized patches of Mylar sandwich films makes the method robust and versatile, allows for storage and shipping of crystals, and enables automated mounting at synchrotrons, as well as goniometer-based screening and data collection. The protocol covers preparation of in situ plates and setup of crystallization trials; 3D printing and assembly of holders; opening of plates, isolation of film patches containing crystals, and loading them onto holders; basic screening and data-collection guidelines; and unloading of holders, as well as reuse and recycling of them. In situ plates are prepared and assembled in 1 h; holders are 3D-printed and assembled in ≤90 min; and an in situ plate is opened, and a film patch containing crystals is isolated and loaded onto a holder in 5 min. Less
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle and are thus drug targets for the treatment of insomnia Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source we determined structures of human orexin receptor in complex with the subtype-selective antagonist EMPA N-ethyl- - -methoxy-pyridin- -yl - toluene- -sulfonyl -amino -N-pyridin- -ylmethyl-acetamide at - and - resolution In comparison with the non-subtype-selective antagonist suvorexant EMPA contacted fewer residues through hydrogen bonds at the orthosteric site explaining the faster dissociation rate Comparisons among these OX R structures in complex with selective antagonists and ... More
Orexin peptides in the brain regulate physiological functions such as the sleep-wake cycle, and are thus drug targets for the treatment of insomnia. Using serial femtosecond crystallography and multi-crystal data collection with a synchrotron light source, we determined structures of human orexin 2 receptor in complex with the subtype-selective antagonist EMPA (N-ethyl-2-[(6-methoxy-pyridin-3-yl)-(toluene-2-sulfonyl)-amino]-N-pyridin-3-ylmethyl-acetamide) at 2.30-Å and 1.96-Å resolution. In comparison with the non-subtype-selective antagonist suvorexant, EMPA contacted fewer residues through hydrogen bonds at the orthosteric site, explaining the faster dissociation rate. Comparisons among these OX2R structures in complex with selective antagonists and previously determined OX1R/OX2R structures bound to non-selective antagonists revealed that the residue at positions 2.61 and 3.33 were critical for the antagonist selectivity in OX2R. The importance of these residues for binding selectivity to OX2R was also revealed by molecular dynamics simulation. These results should facilitate the development of antagonists for orexin receptors. Less
A licensed vaccine for respiratory syncytial virus RSV is unavailable and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants Recently isolated antibodies C and D are substantially more potent than palivizumab and a derivative of D is in clinical trials Here we show that unlike D C preferentially neutralizes subtype A viruses The crystal structure of C bound to the RSV fusion F protein reveals that the overall binding mode of C is similar to that of D but their angles of approach are substantially different Mutagenesis and virological studies demonstrate that RSV F residue is largely ... More
A licensed vaccine for respiratory syncytial virus (RSV) is unavailable, and passive prophylaxis with the antibody palivizumab is restricted to high-risk infants. Recently isolated antibodies 5C4 and D25 are substantially more potent than palivizumab, and a derivative of D25 is in clinical trials. Here we show that unlike D25, 5C4 preferentially neutralizes subtype A viruses. The crystal structure of 5C4 bound to the RSV fusion (F) protein reveals that the overall binding mode of 5C4 is similar to that of D25, but their angles of approach are substantially different. Mutagenesis and virological studies demonstrate that RSV F residue 201 is largely responsible for the subtype specificity of 5C4. These results improve our understanding of subtype-specific immunity and the neutralization breadth requirements of next-generation antibodies, and thereby contribute to the design of broadly protective RSV vaccines. Less
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism signaling and ion homeostasis About half of all drug targets are membrane proteins Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics However the pace of IMP crystallography has been relatively slow due to great challenges in crystallization Lipid cubic phase LCP has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment ... More
Membrane proteins constitute an integral part of biomembrane and play key roles in fundamental biological and physiological processes such as metabolism, signaling, and ion homeostasis. About half of all drug targets are membrane proteins. Elucidation of three-dimensional structures of membrane proteins by X-ray crystallography can provide mechanistic insights for their cellular activity and reveal atomic resolution of architectural details for rational design of novel therapeutics. However, the pace of IMP crystallography has been relatively slow due to great challenges in crystallization. Lipid cubic phase (LCP) has proven to be promising in tackling the crystallization problem by providing a membrane-alike environment. Its bilayer is composed of neutral lipids, such as monoacylglycerols, and can accommodate a substantial amount of native lipids such as phospholipids and cholesterol. Thus, the structure and composition of LCP mimic biomembranes and therefore offer a native-like environment for membrane proteins, which is favorable for functionality and crystallization. Here, the principles for LCP formation, membrane protein reconstitution, and crystallization process are described. The successful application of LCP crystallization for a wide range of membrane proteins including receptors, complexes, transporters, channels, enzymes, membrane protein insertion chaperons, and outer membrane �-barrels is summarized. General methods and protocols for this method are also described. Less
CLYBL encodes a ubiquitously expressed mitochondrial enzyme conserved across all vertebrates whose cellular activity and pathway assignment are unknown Its homozygous loss is tolerated in seemingly healthy individuals with reduced circulating B levels being the only and consistent phenotype reported to date Here by combining enzymology structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells Cells lacking CLYBL accumulate citramalyl-CoA an intermediate in the C -dicarboxylate metabolic pathway that includes itaconate a recently identified human antimicrobial metabolite and immunomodulator We report that CLYBL loss leads to a cell autonomous defect in the ... More
CLYBL encodes a ubiquitously expressed mitochondrial enzyme, conserved across all vertebrates, whose cellular activity and pathway assignment are unknown. Its homozygous loss is tolerated in seemingly healthy individuals, with reduced circulating B12 levels being the only and consistent phenotype reported to date. Here, by combining enzymology, structural biology and activity-based metabolomics we report that CLYBL operates as a citramalyl-CoA lyase in mammalian cells. Cells lacking CLYBL accumulate citramalyl-CoA, an intermediate in the C5-dicarboxylate metabolic pathway that includes itaconate, a recently identified human antimicrobial metabolite and immunomodulator. We report that CLYBL loss leads to a cell autonomous defect in the mitochondrial B12 metabolism and that itaconyl-CoA is a cofactor-inactivating, substrate-analogue inhibitor of the mitochondrial B12-dependent methylmalonyl-CoA mutase (MUT). Our work de-orphans the function of human CLYBL and reveals that a consequence of exposure to the immunomodulatory metabolite itaconate is B12 inactivation. Less
Deubiquitinating enzymes DUBs have garnered significant attention as drug targets in the last years The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins especially proteins that are challenging to directly target but which are stabilized by DUB family members Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools Most reported DUB inhibitors however are polypharmacological agents possessing weak micromolar potency toward their primary target limiting their utility in target validation and mechanism studies Due to a lack of high-resolution DUB small-molecule ligand complex structures no structure-guided ... More
Deubiquitinating enzymes (DUBs) have garnered significant attention as drug targets in the last 5–10 years. The excitement stems in large part from the powerful ability of DUB inhibitors to promote degradation of oncogenic proteins, especially proteins that are challenging to directly target but which are stabilized by DUB family members. Highly optimized and well-characterized DUB inhibitors have thus become highly sought after tools. Most reported DUB inhibitors, however, are polypharmacological agents possessing weak (micromolar) potency toward their primary target, limiting their utility in target validation and mechanism studies. Due to a lack of high-resolution DUB⋅small-molecule ligand complex structures, no structure-guided optimization efforts have been reported for a mammalian DUB. Here, we report a small-molecule⋅ubiquitin-specific protease (USP) family DUB co-structure and rapid design of potent and selective inhibitors of USP7 guided by the structure. Interestingly, the compounds are non-covalent active-site inhibitors. Less
Generation of an electrochemical proton gradient is the first step of cell bioenergetics In prokaryotes the gradient is created by outward membrane protein proton pumps Inward plasma membrane native proton pumps are yet unknown We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins They are inward proton pumps as we demonstrate in model membrane systems Escherichia coli cells human embryonic kidney cells neuroblastoma cells and rat hippocampal neuronal cells We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina NsXeR and suggest a mechanism of inward proton pumping ... More
Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins. Less
NADH and NADPH are redox coenzymes broadly required for energy metabolism biosynthesis and detoxification Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking in part because of a lack of tools with which to investigate them in living cells We previously reported the use of the naturally occurring Lactobacillus brevis H O-forming NADH oxidase LbNOX as a genetic tool for manipulation of the NAD NADH ratio in human cells Here we present TPNOX triphosphopyridine nucleotide oxidase a rationally designed and engineered mutant ... More
NADH and NADPH are redox coenzymes broadly required for energy metabolism, biosynthesis and detoxification. Despite detailed knowledge of specific enzymes and pathways that utilize these coenzymes, a holistic understanding of the regulation and compartmentalization of NADH and NADPH-dependent pathways is lacking, in part because of a lack of tools with which to investigate them in living cells. We previously reported the use of the naturally occurring Lactobacillus brevis H2O-forming NADH oxidase (LbNOX) as a genetic tool for manipulation of the NAD+/NADH ratio in human cells. Here we present TPNOX (triphosphopyridine nucleotide oxidase), a rationally designed and engineered mutant of LbNOX that is strictly specific towards NADPH. We characterize the effects of TPNOX expression on cellular metabolism and use it in combination with LbNOX to show how the redox states of mitochondrial NADPH and NADH pools are connected. Less
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases Given the importance of G protein-coupled receptors GPCRs as pharmaceutical targets there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs Here we present the - resolution structure of a complex between the human -hydroxytryptamine B -HT B receptor and an antibody Fab fragment bound to the extracellular side of the receptor determined by serial femtosecond crystallography with an X-ray free-electron laser The antibody binds to a D epitope of the receptor that includes all three extracellular loops The -HT ... More
Monoclonal antibodies provide an attractive alternative to small-molecule therapies for a wide range of diseases. Given the importance of G protein-coupled receptors (GPCRs) as pharmaceutical targets, there has been an immense interest in developing therapeutic monoclonal antibodies that act on GPCRs. Here we present the 3.0-Å resolution structure of a complex between the human 5-hydroxytryptamine 2B (5-HT2B) receptor and an antibody Fab fragment bound to the extracellular side of the receptor, determined by serial femtosecond crystallography with an X-ray free-electron laser. The antibody binds to a 3D epitope of the receptor that includes all three extracellular loops. The 5-HT2B receptor is captured in a well-defined active-like state, most likely stabilized by the crystal lattice. The structure of the complex sheds light on the mechanism of selectivity in extracellular recognition of GPCRs by monoclonal antibodies. Less
The cannabinoid receptor CB is the principal target of the psychoactive constituent of marijuana the partial agonist -tetrahydrocannabinol -THC Here we report two agonist-bound crystal structures of human CB in complex with a tetrahydrocannabinol AM and a hexahydrocannabinol AM at and resolution respectively The two CB agonist complexes reveal important conformational changes in the overall structure relative to the antagonist-bound state including a reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region In addition a twin toggle switch of Phe and Trp superscripts denote Ballesteros Weinstein numbering is experimentally observed ... More
The cannabinoid receptor 1 (CB1) is the principal target of the psychoactive constituent of marijuana, the partial agonist Δ9-tetrahydrocannabinol (Δ9-THC)1. Here we report two agonist-bound crystal structures of human CB1 in complex with a tetrahydrocannabinol (AM11542) and a hexahydrocannabinol (AM841) at 2.80 Å and 2.95 Å resolution, respectively. The two CB1–agonist complexes reveal important conformational changes in the overall structure, relative to the antagonist-bound state2, including a 53% reduction in the volume of the ligand-binding pocket and an increase in the surface area of the G-protein-binding region. In addition, a ‘twin toggle switch’ of Phe2003.36 and Trp3566.48 (superscripts denote Ballesteros–Weinstein numbering3) is experimentally observed and appears to be essential for receptor activation. The structures reveal important insights into the activation mechanism of CB1 and provide a molecular basis for predicting the binding modes of Δ9-THC, and endogenous and synthetic cannabinoids. The plasticity of the binding pocket of CB1 seems to be a common feature among certain class A G-protein-coupled receptors. These findings should inspire the design of chemically diverse ligands with distinct pharmacological properties. Less
Clinical studies indicate that partial agonists of the G-protein-coupled free fatty acid receptor GPR enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type diabetes mellitus Full allosteric agonists AgoPAMs of GPR bind to a site distinct from partial agonists and can provide additional efficacy We report the - crystal structure of human GPR hGPR in complex with both the partial agonist MK- and an AgoPAM which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle Comparison with an additional - structure of the hGPR MK- binary complex reveals an induced-fit conformational coupling between ... More
Clinical studies indicate that partial agonists of the G-protein-coupled, free fatty acid receptor 1 GPR40 enhance glucose-dependent insulin secretion and represent a potential mechanism for the treatment of type 2 diabetes mellitus. Full allosteric agonists (AgoPAMs) of GPR40 bind to a site distinct from partial agonists and can provide additional efficacy. We report the 3.2-Å crystal structure of human GPR40 (hGPR40) in complex with both the partial agonist MK-8666 and an AgoPAM, which exposes a novel lipid-facing AgoPAM-binding pocket outside the transmembrane helical bundle. Comparison with an additional 2.2-Å structure of the hGPR40–MK-8666 binary complex reveals an induced-fit conformational coupling between the partial agonist and AgoPAM binding sites, involving rearrangements of the transmembrane helices 4 and 5 (TM4 and TM5) and transition of the intracellular loop 2 (ICL2) into a short helix. These conformational changes likely prime GPR40 to a more active-like state and explain the binding cooperativity between these ligands. Less
PP C phosphatases control biological processes including stress responses development and cell division in all kingdoms of life Diverse regulatory domains adapt PP C phosphatases to specific functions but how these domains control phosphatase activity was unknown We present structures representing active and inactive states of the PP C phosphatase SpoIIE from Bacillus subtilis Based on structural analyses and genetic and biochemical experiments we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor Our analysis indicates that this switch is widely conserved among PP C family members serving as a platform ... More
PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from Bacillus subtilis. Based on structural analyses and genetic and biochemical experiments, we identify an a-helical switch that shifts a carbonyl oxygen into the active site to coordinate a metal cofactor. Our analysis indicates that this switch is widely conserved among PP2C family members, serving as a platform to control phosphatase activity in response to diverse inputs. Remarkably, the switch is shared with proteasomal proteases, which we identify as evolutionary and structural relatives of PP2C phosphatases. Although these proteases use an unrelated catalytic mechanism, rotation of equivalent helices controls protease activity by movement of the equivalent carbonyl oxygen into the active site. Less
The Smoothened receptor SMO belongs to the Class Frizzled of the G protein-coupled receptor GPCR superfamily constituting a key component of the Hedgehog signalling pathway Here we report the crystal structure of the multi-domain human SMO bound and stabilized by a designed tool ligand TC using an X-ray free-electron laser source at The structure reveals a precise arrangement of three distinct domains a seven-transmembrane helices domain TMD a hinge domain HD and an intact extracellular cysteine-rich domain CRD This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation By combining the structural data ... More
The Smoothened receptor (SMO) belongs to the Class Frizzled of the G protein-coupled receptor (GPCR) superfamily, constituting a key component of the Hedgehog signalling pathway. Here we report the crystal structure of the multi-domain human SMO, bound and stabilized by a designed tool ligand TC114, using an X-ray free-electron laser source at 2.9 Å. The structure reveals a precise arrangement of three distinct domains: a seven-transmembrane helices domain (TMD), a hinge domain (HD) and an intact extracellular cysteine-rich domain (CRD). This architecture enables allosteric interactions between the domains that are important for ligand recognition and receptor activation. By combining the structural data, molecular dynamics simulation, and hydrogen-deuterium-exchange analysis, we demonstrate that transmembrane helix VI, extracellular loop 3 and the HD play a central role in transmitting the signal employing a unique GPCR activation mechanism, distinct from other multi-domain GPCRs. Less
The glucagon-like peptide- receptor GLP- R and the glucagon receptor GCGR are members of the secretin-like class B family of G-protein-coupled receptors GPCRs and have opposing physiological roles in insulin release and glucose homeostasis The treatment of type diabetes requires positive modulation of GLP- R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner Here we report crystal structures of the human GLP- R transmembrane domain in complex with two different negative allosteric modulators PF- and NNC at and resolution respectively The structures reveal a common binding pocket for negative allosteric modulators present in both GLP- R ... More
The glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are members of the secretin-like class B family of G-protein-coupled receptors (GPCRs) and have opposing physiological roles in insulin release and glucose homeostasis1. The treatment of type 2 diabetes requires positive modulation of GLP-1R to inhibit glucagon secretion and stimulate insulin secretion in a glucose-dependent manner2. Here we report crystal structures of the human GLP-1R transmembrane domain in complex with two different negative allosteric modulators, PF-06372222 and NNC0640, at 2.7 and 3.0 Å resolution, respectively. The structures reveal a common binding pocket for negative allosteric modulators, present in both GLP-1R and GCGR3 and located outside helices V–VII near the intracellular half of the receptor. The receptor is in an inactive conformation with compounds that restrict movement of the intracellular tip of helix VI, a movement that is generally associated with activation mechanisms in class A GPCRs4,5,6. Molecular modelling and mutagenesis studies indicate that agonist positive allosteric modulators target the same general region, but in a distinct sub-pocket at the interface between helices V and VI, which may facilitate the formation of an intracellular binding site that enhances G-protein coupling. Less
We describe a fast easy and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide single-wavelength anomalous diffraction I-SAD The potential universality of the method is based on a common feature of membrane proteins the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins including a human G protein coupled receptor GPCR and we show that I-SAD can be applied using data collection strategies based on either standard or ... More
We describe a fast, easy, and potentially universal method for the de novo solution of the crystal structures of membrane proteins via iodide–single-wavelength anomalous diffraction (I-SAD). The potential universality of the method is based on a common feature of membrane proteins—the availability at the hydrophobic-hydrophilic interface of positively charged amino acid residues with which iodide strongly interacts. We demonstrate the solution using I-SAD of four crystal structures representing different classes of membrane proteins, including a human G protein–coupled receptor (GPCR), and we show that I-SAD can be applied using data collection strategies based on either standard or serial x-ray crystallography techniques. Less
Lipidic cubic phase LCP has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment Application of this material to a wide variety of membrane proteins however is hindered due to a limited number of available host lipids mostly monoacylglycerols MAGs Here we designed synthesized and characterized a series of chemically stable lipids resistant to hydrolysis with properties complementary to the widely used MAGs In order to assess their potential to serve as host lipids for crystallization we characterized the phase properties and lattice parameters of mesophases made of ... More
Lipidic cubic phase (LCP) has been widely recognized as a promising membrane-mimicking matrix for biophysical studies of membrane proteins and their crystallization in a lipidic environment. Application of this material to a wide variety of membrane proteins, however, is hindered due to a limited number of available host lipids, mostly monoacylglycerols (MAGs). Here, we designed, synthesized and characterized a series of chemically stable lipids resistant to hydrolysis, with properties complementary to the widely used MAGs. In order to assess their potential to serve as host lipids for crystallization, we characterized the phase properties and lattice parameters of mesophases made of two most promising lipids at a variety of different conditions by polarized light microscopy and small-angle X-ray scattering. Both lipids showed remarkable chemical stability and an extended LCP region in the phase diagram covering a wide range of temperatures down to 4 °C. One of these lipids has been used for crystallization and structure determination of a prototypical membrane protein bacteriorhodopsin at 4 °C and 20 °C. Less
Angiotensin II receptors AT R and AT R serve as key components of the renin-angiotensin-aldosterone system While AT R plays a central role in the regulation of blood pressure the function of AT R is enigmatic with a variety of reported effects To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors we report crystal structures of the human AT R bound to an AT R-selective and an AT R AT R-dual ligand respectively capturing the receptor in an active-like conformation Unexpectedly helix VIII was found in a non-canonical position stabilizing the active-like state but at ... More
Angiotensin II receptors, AT1R and AT2R, serve as key components of the renin-angiotensin-aldosterone system. While AT1R plays a central role in the regulation of blood pressure, the function of AT2R is enigmatic with a variety of reported effects. To elucidate the mechanisms for the functional diversity and ligand selectivity between these receptors, we report crystal structures of the human AT2R bound to an AT2R-selective and an AT1R/AT2R-dual ligand, respectively, capturing the receptor in an active-like conformation. Unexpectedly, helix VIII was found in a non-canonical position, stabilizing the active-like state, but at the same time preventing the recruitment of G proteins/β-arrestins, in agreement with the lack of signaling responses in standard cellular assays. Structure-activity relationship, docking and mutagenesis studies revealed the interactions critical for ligand binding and selectivity. Our results thus provide insights into the structural basis for distinct functions of the angiotensin receptors, and may guide the design of novel selective ligands. Less
The Haemophilus surface fibril Hsf is an unusually large trimeric autotransporter adhesin TAA expressed by the most virulent strains of H influenzae Hsf is known to mediate adhesion between pathogen and host allowing the establishment of potentially deadly diseases such as epiglottitis meningitis and pneumonia While recent research has suggested that this TAA might adopt a novel hairpin-like architecture the characterization of Hsf has been limited to in silico modelling and electron micrographs with no high-resolution structural data available Here the crystal structure of Hsf putative domain PD is reported at resolution The structure corrects the previous domain annotation by ... More
The Haemophilus surface fibril (Hsf) is an unusually large trimeric autotransporter adhesin (TAA) expressed by the most virulent strains of H. influenzae. Hsf is known to mediate adhesion between pathogen and host, allowing the establishment of potentially deadly diseases such as epiglottitis, meningitis and pneumonia. While recent research has suggested that this TAA might adopt a novel ‘hairpin-like’ architecture, the characterization of Hsf has been limited to in silico modelling and electron micrographs, with no high-resolution structural data available. Here, the crystal structure of Hsf putative domain 1 (PD1) is reported at 3.3 Å resolution. The structure corrects the previous domain annotation by revealing the presence of an unexpected N-terminal TrpRing domain. PD1 represents the first Hsf domain to be solved, and thus paves the way for further research on the ‘hairpin-like’ hypothesis. Less
Membrane-like nanodiscs ND have become an important tool for the cell-free expression solubilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs embedded in NDs would be of high importance for structural biology However despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography In the present work we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso Bacteriorhodopsin BR reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The resulting crystals ... More
Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 Å. The structure of BR was solved at 1.9 Å and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs. Less
Cannabinoid receptor CB is the principal target of -tetrahydrocannabinol THC a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use CB is activated by endocannabinoids and is a promising therapeutic target for pain management inflammation obesity and substance abuse disorders Here we present the crystal structure of human CB in complex with AM a stabilizing antagonist synthesized and characterized for this structural study The structure of the CB -AM complex reveals key features of the receptor and critical interactions for antagonist binding In combination with functional studies and molecular modeling ... More
Cannabinoid receptor 1 (CB1) is the principal target of ?9-tetrahydrocannabinol (THC), a psychoactive chemical from Cannabis sativa with a wide range of therapeutic applications and a long history of recreational use. CB1 is activated by endocannabinoids and is a promising therapeutic target for pain management, inflammation, obesity, and substance abuse disorders. Here, we present the 2.8 � crystal structure of human CB1 in complex with AM6538, a stabilizing antagonist, synthesized and characterized for this structural study. The structure of the CB1-AM6538 complex reveals key features of the receptor and critical interactions for antagonist binding. In combination with functional studies and molecular modeling, the structure provides insight into the binding mode of naturally occurring CB1 ligands, such as THC, and synthetic cannabinoids. This enhances our understanding of the molecular basis for the physiological functions of CB1 and provides new opportunities for the design of next-generation CB1-targeting pharmaceuticals. Less
Human metapneumovirus hMPV is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age The hMPV fusion F glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen To facilitate structure-based vaccine design we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of by X-ray crystallography The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus hRSV In contrast significant differences were apparent ... More
Human metapneumovirus (hMPV) is a paramyxovirus that is a common cause of bronchiolitis and pneumonia in children less than five years of age. The hMPV fusion (F) glycoprotein is the primary target of neutralizing antibodies and is thus a critical vaccine antigen. To facilitate structure-based vaccine design, we stabilized the ectodomain of the hMPV F protein in the postfusion conformation and determined its structure to a resolution of 3.3 Å by X-ray crystallography. The structure resembles an elongated cone and is very similar to the postfusion F protein from the related human respiratory syncytial virus (hRSV). In contrast, significant differences were apparent with the postfusion F proteins from other paramyxoviruses, such as human parainfluenza type 3 (hPIV3) and Newcastle disease virus (NDV). The high similarity of hMPV and hRSV postfusion F in two antigenic sites targeted by neutralizing antibodies prompted us to test for antibody cross-reactivity. The widely used monoclonal antibody 101F, which binds to antigenic site IV of hRSV F, was found to cross-react with hMPV postfusion F and neutralize both hRSV and hMPV. Despite the cross-reactivity of 101F and the reported cross-reactivity of two other antibodies, 54G10 and MPE8, we found no detectable cross-reactivity in the polyclonal antibody responses raised in mice against the postfusion forms of either hMPV or hRSV F. The postfusion-stabilized hMPV F protein did, however, elicit high titers of hMPV-neutralizing activity, suggesting that it could serve as an effective subunit vaccine. Structural insights from these studies should be useful for designing novel immunogens able to induce wider cross-reactive antibody responses. Less
Serial femtosecond X-ray crystallography SFX using an X-ray free electron laser XFEL is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins including G-protein coupled receptors GPCRs which often only produce microcrystals An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in Here we report the deposition of the XFEL data and provide further details on crystallization XFEL data collection and analysis structure determination and the validation of the structural model The rhodopsin-arrestin crystal structure solved with ... More
Serial femtosecond X-ray crystallography (SFX) using an X-ray free electron laser (XFEL) is a recent advancement in structural biology for solving crystal structures of challenging membrane proteins, including G-protein coupled receptors (GPCRs), which often only produce microcrystals. An XFEL delivers highly intense X-ray pulses of femtosecond duration short enough to enable the collection of single diffraction images before significant radiation damage to crystals sets in. Here we report the deposition of the XFEL data and provide further details on crystallization, XFEL data collection and analysis, structure determination, and the validation of the structural model. The rhodopsin-arrestin crystal structure solved with SFX represents the first near-atomic resolution structure of a GPCR-arrestin complex, provides structural insights into understanding of arrestin-mediated GPCR signaling, and demonstrates the great potential of this SFX-XFEL technology for accelerating crystal structure determination of challenging proteins and protein complexes. Less
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology In the case of G protein-coupled receptors GPCRs we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis The nociceptin orphanin FQ peptide receptor NOP is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening We observed that antagonist potency is correlated with a ligand s ability to induce receptor stability Tm and crystallogenesis Using this screening strategy we solved two structures of NOP in complex ... More
Understanding the mechanism by which ligands impact receptor conformational equilibria is key in accelerating membrane protein structural biology. In the case of G protein-coupled receptors (GPCRs) we currently pursue a brute force approach for identifying ligands that stabilize receptors and facilitate crystallogenesis. The nociceptin/orphanin FQ peptide receptor (NOP) is a member of the opioid receptor subfamily of GPCRs for which many structurally diverse ligands are available for screening. We observed that antagonist potency is correlated with a ligand’s ability to induce receptor stability (Tm) and crystallogenesis. Using this screening strategy, we solved two structures of NOP in complex with top candidate ligands SB-612111 and C-35. Docking studies indicate that while potent, stabilizing antagonists strongly favor a single binding orientation, less potent ligands can adopt multiple binding modes, contributing to their low Tm values. These results suggest a mechanism for ligand-aided crystallogenesis whereby potent antagonists stabilize a single ligand-receptor conformational pair. Less
Angiotensin II type receptor AT R is the primary blood pressure regulator AT R blockers ARBs have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies The structural basis for AT R modulation by different peptide and non-peptide ligands has remained elusive Here we report the crystal structure of the human AT R in complex with an inverse agonist olmesartan BenicarTM a highly potent anti-hypertensive drug Olmesartan is anchored to the receptor primarily by the residues Tyr- Trp- and Arg- ECL ... More
Angiotensin II type 1 receptor (AT1R) is the primary blood pressure regulator. AT1R blockers (ARBs) have been widely used in clinical settings as anti-hypertensive drugs and share a similar chemical scaffold, although even minor variations can lead to distinct therapeutic efficacies toward cardiovascular etiologies. The structural basis for AT1R modulation by different peptide and non-peptide ligands has remained elusive. Here, we report the crystal structure of the human AT1R in complex with an inverse agonist olmesartan (BenicarTM), a highly potent anti-hypertensive drug. Olmesartan is anchored to the receptor primarily by the residues Tyr-351.39, Trp-842.60, and Arg-167ECL2, similar to the antagonist ZD7155, corroborating a common binding mode of different ARBs. Using docking simulations and site-directed mutagenesis, we identified specific interactions between AT1R and different ARBs, including olmesartan derivatives with inverse agonist, neutral antagonist, or agonist activities. We further observed that the mutation N1113.35A in the putative sodium-binding site affects binding of the endogenous peptide agonist angiotensin II but not the β-arrestin-biased peptide TRV120027. Less
A number of Gram-positive bacteria produce a class of bacteriocins called lantibiotics These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria including clinically challenging pathogens and are therefore potential alternatives to antibiotics All lantibiotic producer strains and some Gram-positive non producer strains express protein systems to circumvent a suicidal effect or to become resistant respectively Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins One of the best-characterized lantibiotics is nisin which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria including some ... More
A number of Gram-positive bacteria produce a class of bacteriocins called ‘lantibiotics’. These lantibiotics are ribosomally synthesized peptides that possess high antimicrobial activity against Gram-positive bacteria, including clinically challenging pathogens, and are therefore potential alternatives to antibiotics. All lantibiotic producer strains and some Gram-positive nonproducer strains express protein systems to circumvent a suicidal effect or to become resistant, respectively. Two-component systems consisting of a response regulator and a histidine kinase upregulate the expression of these proteins. One of the best-characterized lantibiotics is nisin, which is produced by Lactococcus lactis and possesses bactericidal activity against various Gram-positive bacteria, including some human pathogenic strains. Within many human pathogenic bacterial strains inherently resistant to nisin, a response regulator, NsrR, has been identified which regulates the expression of proteins involved in nisin resistance. In the present study, an expression and purification protocol was established for the NsrR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method, resulting in crystals that diffracted X-rays to 1.4 Å resolution. Less
G protein-coupled receptors GPCRs signal primarily through G proteins or arrestins Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin determined by serial femtosecond X-ray laser crystallography Together with extensive biochemical and mutagenesis data the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements including TM and Helix to recruit arrestin Correspondingly arrestin adopts the pre-activated conformation with a rotation between the ... More
G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the N- and C- domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signaling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology. Less
Macromolecular X-ray crystallography has been the primary methodology for determining the three-dimensional structures of proteins nucleic acids and viruses Structural information has paved the way for structure-guided drug discovery and laid the foundations for structural bioinformatics However X-ray crystallography still has a few fundamental limitations some of which may be overcome and complemented using emerging methods and technologies in other areas of structural biology
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology Bacteriorhodopsin from Halobium salinarum bR is a striking example of the difficulties in membrane protein overexpression We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression This complementary protein approach allowed us to increase bR expression by two orders of magnitude ... More
Heterologous overexpression of functional membrane proteins is a major bottleneck of structural biology. Bacteriorhodopsin from Halobium salinarum (bR) is a striking example of the difficulties in membrane protein overexpression. We suggest a general approach with a finite number of steps which allows one to localize the underlying problem of poor expression of a membrane protein using bR as an example. Our approach is based on constructing chimeric proteins comprising parts of a protein of interest and complementary parts of a homologous protein demonstrating advantageous expression. This complementary protein approach allowed us to increase bR expression by two orders of magnitude through the introduction of two silent mutations into bR coding DNA. For the first time the high quality crystals of bR expressed in E. Coli were obtained using the produced protein. The crystals obtained with in meso nanovolume crystallization diffracted to 1.67 Å. Less
Nisin is a -amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics Nisin displays a high bactericidal activity against various Gram-positive bacteria including some human-pathogenic strains However there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome The encoded protein NSR cleaves off the last six amino acids of nisin thereby reducing its bactericidal efficacy An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops resulting ... More
Nisin is a 34-amino-acid antimicrobial peptide produced by Lactococcus lactis belonging to the class of lantibiotics. Nisin displays a high bactericidal activity against various Gram-positive bacteria, including some human-pathogenic strains. However, there are some nisin-non-producing strains that are naturally resistant owing to the presence of the nsr gene within their genome. The encoded protein, NSR, cleaves off the last six amino acids of nisin, thereby reducing its bactericidal efficacy. An expression and purification protocol has been established for the NSR protein from Streptococcus agalactiae COH1. The protein was successfully crystallized using the vapour-diffusion method in hanging and sitting drops, resulting in crystals that diffracted X-rays to 2.8 and 2.2 Å, respectively. Less
Angiotensin II type receptor AT R is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance Although several anti-hypertensive drugs have been developed as AT R blockers ARBs the structural basis for AT R ligand-binding and regulation has remained elusive mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser we successfully determined the room-temperature crystal structure of the human AT R in complex with its selective antagonist ZD at resolution The AT R-ZD ... More
Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9 � resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design. Less
Recently the first known light-driven sodium pumps from the microbial rhodopsin family were discovered We have solved the structure of one of them Krokinobacter eikastus rhodopsin KR in the monomeric blue state and in two pentameric red states at resolutions of and and respectively The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface that the ion-release cavity is capped by a unique N-terminal -helix and that the ion-uptake cavity is unexpectedly large and open to the surface Obstruction of the cavity with the mutation G F imparts KR ... More
Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 Å and 2.2 and 2.8 Å, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics. Less
Crystallization of integral membrane proteins MPs is notoriously difficult given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process MP crystallization in a membrane mimetic matrix known as lipidic cubic phase LCP has recently started to gain popularity following successes in structure determination of G protein-coupled receptors GPCRs transporters and enzymes Unlike crystallization trials in aqueous solutions where protein molecules are free to move diffusion of MPs in LCP is restricted and thus a high level of protein mobility can serve as an early indication for subsequent crystallization success Prompted by ... More
Crystallization of integral membrane proteins (MPs) is notoriously difficult, given their poor stability outside native membrane environment and due to the interference of detergent micelles with crystallization process. MP crystallization in a membrane mimetic matrix, known as lipidic cubic phase (LCP), has recently started to gain popularity, following successes in structure determination of G protein-coupled receptors (GPCRs), transporters, and enzymes. Unlike crystallization trials in aqueous solutions where protein molecules are free to move, diffusion of MPs in LCP is restricted, and, thus, a high level of protein mobility can serve as an early indication for subsequent crystallization success. Prompted by our initial observations that precipitant conditions can dramatically affect diffusion of GPCRs in LCP, we have developed a simple precrystallization assay, based on measuring protein diffusion at a number of different conditions by fluorescence recovery after photobleaching (LCP-FRAP). Over the last few years, the LCP-FRAP assay was incorporated in our GPCR structure determination pipeline and proved as a powerful technique allowing for a faster identification of crystallization conditions for many different receptors. The assay is used to screen for the best protein constructs, ligands, LCP host lipids, precipitants, and additives, thereby focusing subsequent crystallization trials on the most promising parts of the multidimensional crystallization phase diagram, substantially increasing the likelihood of finding the right crystallization condition. Here, we describe our LCP-FRAP protocols for guiding GPCR crystallization, which can be adapted to any other MP, and discuss some of the critical considerations related to application of this assay. Less
Na -translocating NADH quinone oxidoreductase NQR is a redox-driven sodium pump operating in the respiratory chain of various bacteria including pathogenic species The enzyme has a unique set of redox active prosthetic groups which includes two covalently bound flavin mononucleotide FMN residues attached to threonine residues in subunits NqrB and NqrC The reason of FMN covalent bonding in the subunits has not been established yet In the current work binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding To study ... More
Na+-translocating NADH:quinone oxidoreductase (NQR) is a redox-driven sodium pump operating in the respiratory chain of various bacteria, including pathogenic species. The enzyme has a unique set of redox active prosthetic groups, which includes two covalently bound flavin mononucleotide (FMN) residues attached to threonine residues in subunits NqrB and NqrC. The reason of FMN covalent bonding in the subunits has not been established yet. In the current work, binding of free FMN to the apo-form of NqrC from Vibrio harveyi was studied showing very low affinity of NqrC to FMN in the absence of its covalent bonding. To study structural aspects of flavin binding in NqrC, its holo-form was crystallized and its 3D structure was solved at 1.56 Å resolution. It was found that the isoalloxazine moiety of the FMN residue is buried in a hydrophobic cavity and that its pyrimidine ring is squeezed between hydrophobic amino acid residues while its benzene ring is extended from the protein surroundings. This structure of the flavin-binding pocket appears to provide flexibility of the benzene ring, which can help the FMN residue to take the bended conformation and thus to stabilize the one-electron reduced form of the prosthetic group. These properties may also lead to relatively weak noncovalent binding of the flavin. This fact along with periplasmic location of the FMN-binding domains in the vast majority of NqrC-like proteins may explain the necessity of the covalent bonding of this prosthetic group to prevent its loss to the external medium. Less
Bi-functional - and - opioid receptor OR ligands are potential therapeutic alternatives to alkaloid opiate analgesics with diminished side effects We solved the structure of human -OR bound to the bi-functional -OR antagonist and -OR agonist tetrapeptide H-Dmt -Tic -Phe -Phe -NH DIPP-NH by serial femtosecond crystallography revealing a cis-peptide bond between H-Dmt and Tic The observed receptor-peptide interactions are critical to understand the pharmacological profiles of opioid peptides and to develop improved analgesics
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps Here we present the crystal structure of a new member of the family Haloarcula marismortui bacteriorhodopsin I HmBRI D N mutant at the resolution of While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps its proton release region is extended and contains additional water molecules The protein's fold is reinforced by three novel inter-helical hydrogen bonds two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins Despite the expression in ... More
Bacteriorhodopsins are a large family of seven-helical transmembrane proteins that function as light-driven proton pumps. Here, we present the crystal structure of a new member of the family, Haloarcula marismortui bacteriorhodopsin I (HmBRI) D94N mutant, at the resolution of 2.5 Å. While the HmBRI retinal-binding pocket and proton donor site are similar to those of other archaeal proton pumps, its proton release region is extended and contains additional water molecules. The protein's fold is reinforced by three novel inter-helical hydrogen bonds, two of which result from double substitutions relative to Halobacterium salinarum bacteriorhodopsin and other similar proteins. Despite the expression in Escherichia coli and consequent absence of native lipids, the protein assembles as a trimer in crystals. The unique extended loop between the helices D and E of HmBRI makes contacts with the adjacent protomer and appears to stabilize the interface. Many lipidic hydrophobic tail groups are discernible in the membrane region, and their positions are similar to those of archaeal isoprenoid lipids in the crystals of other proton pumps, isolated from native or native-like sources. All these features might explain the HmBRI properties and establish the protein as a novel model for the microbial rhodopsin proton pumping studies. Less
Amphipols APols have become important tools for the stabilization folding and in vitro structural and functional studies of membrane proteins MPs Direct crystallization of MPs solubilized in APols would be of high importance for structural biology However despite considerable efforts it is still not clear whether MP APol complexes can form well-ordered crystals suitable for X-ray crystallography In the present work we show that an APol-trapped MP can be crystallized in meso Bacteriorhodopsin BR trapped by APol A - was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant The crystals diffract beyond The structure of ... More
Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs. Less
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase LCP-SFX for protein structure determination at X-ray free electron lasers XFELs LCP-SFX uses the gel-like lipidic cubic phase LCP as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection LCP is a liquid-crystalline mesophase composed of lipids and water It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room ... More
We have recently established a procedure for serial femtosecond crystallography in lipidic cubic phase (LCP-SFX) for protein structure determination at X-ray free electron lasers (XFELs). LCP-SFX uses the gel-like lipidic cubic phase (LCP) as a matrix for growth and delivery of membrane protein microcrystals for crystallographic data collection. LCP is a liquid-crystalline mesophase, composed of lipids and water. It provides a membrane-mimicking environment that stabilizes membrane proteins and supports their crystallization. Here we describe detailed procedures for the preparation and characterization of microcrystals for LCP-SFX applications. The advantages of LCP-SFX over traditional crystallographic methods include the capability of collecting room temperature high-resolution data with minimal effects of radiation damage from sub-10 �m crystals of membrane and soluble proteins that are difficult to crystallize, while eliminating the need for crystal harvesting and cryo-cooling. Compared to SFX methods for microcrystals in solution using liquid injectors, LCP-SFX reduces protein consumption by 2�3 orders of magnitude for data collection at currently available XFELs. The whole procedure typically takes 3�5 days, including the time required for crystals to grow. Less
The Smoothened receptor SMO mediates signal transduction in the hedgehog pathway which is implicated in normal development and carcinogenesis SMO antagonists can suppress the growth of some tumors however mutations at SMO have been found to abolish their anti-tumor effects a phenomenon known as chemoresistance Here we report three crystal structures of human SMO bound to the antagonists SANT and Anta XV and the agonist SAG at resolution The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites where SANT binds at a deeper site as compared with other ligands Distinct interactions at D ... More
The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumors; however, mutations at SMO have been found to abolish their anti-tumor effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6–2.8Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbors multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D4736.55 elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules. Less
Lipidic cubic phase LCP is a gel-like liquid crystalline membrane-mimetic matrix It has been successfully used to stabilize and crystallize challenging membrane proteins such as G protein-coupled receptors the structure of which is often difficult to obtain by other methods Despite many advantages the LCP crystallization method has not been widely adopted because of difficulties associated with handling highly viscous LCP material Recent advances in the development of tools and instruments for LCP crystallization are aimed at facilitating the research in this area and to help structural biologists in integrating these technologies in their working routine
Phospholipids have major roles in the structure and function of all cell membranes Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain IPCT fused with a membrane CDP-alcohol phosphotransferase domain DIPPS at resolution The bifunctional protein dimerizes through the DIPPS domains each comprising six transmembrane -helices The active site cavity is hydrophilic and widely ... More
Phospholipids have major roles in the structure and function of all cell membranes. Most integral membrane proteins from the large CDP-alcohol phosphatidyltransferase family are involved in phospholipid biosynthesis across the three domains of life. They share a conserved sequence pattern and catalyse the displacement of CMP from a CDP-alcohol by a second alcohol. Here we report the crystal structure of a bifunctional enzyme comprising a cytoplasmic nucleotidyltransferase domain (IPCT) fused with a membrane CDP-alcohol phosphotransferase domain (DIPPS) at 2.65 Å resolution. The bifunctional protein dimerizes through the DIPPS domains, each comprising six transmembrane α-helices. The active site cavity is hydrophilic and widely open to the cytoplasm with a magnesium ion surrounded by four highly conserved aspartate residues from helices TM2 and TM3. We show that magnesium is essential for the enzymatic activity and is involved in catalysis. Substrates docking is validated by mutagenesis studies, and a structure-based catalytic mechanism is proposed. Less
Lipidic cubic phase LCP crystallization has proven successful for high-resolution structure determination of challenging membrane proteins Here we present a technique for extruding gel-like LCP with embedded membrane protein microcrystals providing a continuously renewed source of material for serial femtosecond crystallography Data collected from sub- - m-sized crystals produced with less than mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor
Opioids represent widely prescribed and abused medications although their signal transduction mechanisms are not well understood Here we present the high-resolution crystal structure of the human -opioid receptor -OR revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity The distinctive -OR sodium ion site architecture is centrally located in a polar interaction network in the -transmembrane bundle core with the sodium ion stabilizing a reduced agonist affinity state and thereby modulating signal transduction Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn to alanine ... More
Opioids represent widely prescribed and abused medications, although their signal transduction mechanisms are not well understood. Here we present the 1.8Å high-resolution crystal structure of the human δ-opioid receptor (δ-OR), revealing the presence and fundamental role of a sodium ion mediating allosteric control of receptor functional selectivity and constitutive activity. The distinctive δ-OR sodium ion site architecture is centrally located in a polar interaction network in the 7-transmembrane bundle core, with the sodium ion stabilizing a reduced agonist affinity state, and thereby modulating signal transduction. Site-directed mutagenesis and functional studies reveal that changing the allosteric sodium site residue Asn131 to alanine or valine augments constitutive arrestin-ergic signaling. Asp95Ala, Asn310Ala, and Asn314Ala mutations transform classical δ-opioid antagonists like naltrindole into potent β-arrestin-biased agonists. The data establish the molecular basis for allosteric sodium ion control in opioid signaling, revealing that sodium-coordinating residues act as “efficacy-switches” at a prototypic G protein-coupled receptor. Less
Nonlinear optical NLO instrumentation has been integrated with synchrotron X-ray diffraction XRD for combined single-platform analysis initially targeting applications for automated crystal centering Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning Two optical designs were constructed and characterized one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer Both instruments enabled protein crystal identification with integration times between and s per pixel representing a -fold reduction in the per-pixel exposure time relative to X-ray raster scanning Quantitative centering and analysis of phenylalanine hydroxylase ... More
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied. Less
Introduction X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution It provides key information for the understanding of protein function opening opportunities for the modulation of enzymatic mechanisms and protein ligand interactions As a consequence macromolecular crystallography plays an essential role in drug design as well as in the a posteriori validation of drug mechanisms Areas covered The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past years As a consequence access to the facilities required for these investigations such as synchrotron beamlines became more difficult and significant efforts ... More
Introduction: X-ray crystallography is the main tool for macromolecular structure solution at atomic resolution. It provides key information for the understanding of protein function, opening opportunities for the modulation of enzymatic mechanisms, and protein�ligand interactions. As a consequence, macromolecular crystallography plays an essential role in drug design, as well as in the a posteriori validation of drug mechanisms.
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
Areas covered: The demand for method developments and also tools for macromolecular crystallography has significantly increased over the past 10 years. As a consequence, access to the facilities required for these investigations, such as synchrotron beamlines, became more difficult and significant efforts were dedicated to the automation of the experimental setup in laboratories. In this article, the authors describe how this was accomplished and how robot-based systems contribute to the enhancement of the macromolecular structure solution pipeline.
Expert opinion: The evolution in robot technology, together with progress in X-ray beam performance and software developments, contributes to a new era in macromolecular X-ray crystallography. Highly integrated experimental environments open new possibilities for crystallography experiments. It is likely that it will also change the way this technique will be used in the future, opening the field to a larger community. Less
The smoothened SMO receptor a key signal transducer in the Hedgehog Hh signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis The SMO receptor is classified as a class Frizzled class F G protein-coupled receptor GPCR although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than Here we report the crystal structure at resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY Although the SMO receptor shares the seven transmembrane helical TM fold ... More
The smoothened (SMO) receptor, a key signal transducer in the Hedgehog (Hh) signaling pathway is both responsible for the maintenance of normal embryonic development and implicated in carcinogenesis. The SMO receptor is classified as a class Frizzled (class F) G protein-coupled receptor (GPCR), although the canonical Hh signaling pathway involves the transcription factor Gli and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure at 2.5 Å resolution of the transmembrane domain of the human SMO receptor bound to the small molecule antagonist LY2940680. Although the SMO receptor shares the seven transmembrane helical (7TM) fold, most conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulfide bonds. The ligand binds at the extracellular end of the 7TM bundle and forms extensive contacts with the loops. Less
Structural studies of human G protein-coupled receptors GPCRs have recently been accelerated through the use of the T lysozyme fusion partner that was inserted into the third intracellular loop Using chimeras of the human -adrenergic and human A A adenosine receptors we present the methodology and data for the selection of five new fusion partners for crystallizing GPCRs In particular the use of the thermostabilized apocytochrome b RIL as a fusion partner displays certain advantages over the previously utilized T lysozyme resulting in a significant improvement in stability and structure in GPCR-fusion constructs
Members of the opioid receptor family of G-protein-coupled receptors GPCRs are found throughout the peripheral and central nervous system where they have key roles in nociception and analgesia Unlike the classical opioid receptors and -OR -OR and -OR which were delineated by pharmacological criteria in the s and s the nociceptin orphanin FQ N OFQ peptide receptor NOP also known as ORL- was discovered relatively recently by molecular cloning and characterization of an orphan GPCR Although it shares high sequence similarity with classical opioid GPCR subtypes NOP has a markedly distinct pharmacology featuring activation by the endogenous peptide N OFQ ... More
Members of the opioid receptor family of G-protein-coupled receptors (GPCRs) are found throughout the peripheral and central nervous system, where they have key roles in nociception and analgesia. Unlike the ‘classical’ opioid receptors, δ, κ and μ (δ-OR, κ-OR and μ-OR), which were delineated by pharmacological criteria in the 1970s and 1980s, the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP, also known as ORL-1) was discovered relatively recently by molecular cloning and characterization of an orphan GPCR1. Although it shares high sequence similarity with classical opioid GPCR subtypes (∼60%), NOP has a markedly distinct pharmacology, featuring activation by the endogenous peptide N/OFQ, and unique selectivity for exogenous ligands2,3. Here we report the crystal structure of human NOP, solved in complex with the peptide mimetic antagonist compound-24 (C-24) (ref. 4), revealing atomic details of ligand–receptor recognition and selectivity. Compound-24 mimics the first four amino-terminal residues of the NOP-selective peptide antagonist UFP-101, a close derivative of N/OFQ, and provides important clues to the binding of these peptides. The X-ray structure also shows substantial conformational differences in the pocket regions between NOP and the classical opioid receptors κ (ref. 5) and μ (ref. 6), and these are probably due to a small number of residues that vary between these receptors. The NOP–compound-24 structure explains the divergent selectivity profile of NOP and provides a new structural template for the design of NOP ligands. Less
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps This has enabled the crystallization of large numbers of proteins including integral membrane proteins This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality The latter underscores the importance of the ... More
Structure determination of biological macromolecules using x-ray crystallography has been greatly improved in recent years through the development of a number of key technologies and better integration with sample preparation steps. This has enabled the crystallization of large numbers of proteins, including integral membrane proteins. This chapter discusses the crystallization process as well as the development of automation that has dramatically increased the likelihood of success. Most noteworthy has been the introduction of crystallization protocols that use nanoliter protein solutions and the insight that production of high-quality crystals requires high protein sample quality. The latter underscores the importance of the development of powerful biophysical characterization techniques. Less
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes including the regulation of pain respiratory drive mood and in the case of -opioid receptor -OR dysphoria and psychotomimesis Here we report the crystal structure of the human -OR in complex with the selective antagonist JDTic arranged in parallel dimers at resolution The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human -OR Modelling of other important -OR-selective ligands including the morphinan-derived antagonists norbinaltorphimine and -guanidinonaltrindole and the diterpene agonist salvinorin A analogue ... More
Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and—in the case of κ-opioid receptor (κ-OR)—dysphoria and psychotomimesis. Here we report the crystal structure of the human κ-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 Å resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human κ-OR. Modelling of other important κ-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5′-guanidinonaltrindole, and the diterpene agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure–activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for κ-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human κ-OR. Less
Lipidic cubic phase LCP is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment LCP technologies however have not been fully embraced by the membrane protein structural biology community primarily because of the difficulties associated with handling viscous materials Recent developments of pre-crystallization assays and improvements in crystal imaging successes in obtaining high resolution structures of G protein-coupled receptors GPCRs and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein ... More
Lipidic cubic phase (LCP) is a membrane-mimetic matrix suitable for stabilization and crystallization of membrane proteins in lipidic environment. LCP technologies, however, have not been fully embraced by the membrane protein structural biology community, primarily because of the difficulties associated with handling viscous materials. Recent developments of pre-crystallization assays and improvements in crystal imaging, successes in obtaining high resolution structures of G protein-coupled receptors (GPCRs), and commercial availability of LCP tools and instruments are beginning to attract structural biologists to integrate LCP technologies in their research. This wider acceptance should translate to an increased number of otherwise difficult-to-crystallize membrane protein structures, shedding light on their functional mechanisms and on structural details of lipid-protein interactions. Less