315 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 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