419 Citations
The receptor binding domain of spike protein of SARS-CoV- binds angitensin coverting enzyme on the surface of epithelial cells leading to fusion and entry of virus into the cell Based on our experience we described a work flow for expressing and purifying proteins and screening conditions for generating diffraction quality crystals of the complex Production and crystallization of protein complex take abut twelve days from construction of vectors to harvesting and freezing crystals for data collection
De novo-designed receptor transmembrane domains TMDs present opportunities for precise control of cellular receptor functions We developed a de novo design strategy for generating programmed membrane proteins proMPs single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor CAR that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD from monomers up to tetramers All programmed CARs stimulated substantially ... More
De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology. Less
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications Bilirubin oxidase BOD is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials BOD has been extensively investigated over the last few decades While the enzyme s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated a crystal structure of BOD from bacterial origin has never been determined Here we present the first crystal structure of BOD from Bacillus pumilus BpBOD at resolution Overall BpBOD shows high homology with the fungal enzymes however it holds a ... More
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. Less
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents We recently developed KD a fully human antibody targeting vascular endothelial growth factor receptor VEGFR KD blocked VEGF-A and VEGF-C-mediated VEGFR activation as demonstrated by the in vitro binding and competition assays and functional cellular assays Here we report a computational model of the complex between the variable fragment of KD KD Fv and the domains and of the extracellular portion of VEGFR VEGFR D - Our modeling was guided by a priori experimental information including ... More
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents. We recently developed KD035, a fully human antibody targeting vascular endothelial growth factor receptor 2 (VEGFR2). KD035 blocked VEGF-A, and VEGF-C-mediated VEGFR2 activation, as demonstrated by the in vitro binding and competition assays and functional cellular assays. Here, we report a computational model of the complex between the variable fragment of KD035 (KD035(Fv)) and the domains 2 and 3 of the extracellular portion of VEGFR2 (VEGFR2(D2-3)). Our modeling was guided by a priori experimental information including the X-ray structures of KD035 and related antibodies, binding assays, target domain mapping and comparison of KD035 affinity for VEGFR2 from different species. The accuracy of the model was assessed by molecular dynamics simulations, and subsequently validated by mutagenesis and binding analysis. Importantly, the steps followed during the generation of this model can set a precedent for future in silico efforts aimed at the accurate description of the antibody–antigen and more broadly protein–protein complexes. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper center coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in term of copper-binding ligands copper center geometry redox potential as well as biological function AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features highly resistant to acidic pH it possesses a constrained green-type copper center of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network. Less
Over the last two decades fragment-based drug discovery FBDD has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods However it is also often used with great success as a primary screening technique In recent years technological advances at macromolecular crystallo graphy beamlines in terms of instrumentation beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography Here the development of ... More
Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported. Less
The human chemokines CCL and CCL bind to the G protein-coupled receptor GPCR CCR and play an important role in the trafficking of immune cells as well as cancer metastasis Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine GPCR interaction and consequently tumor metastasis Here we report the first X-ray crystal structure of a truncated CCL residues at resolution revealing molecular details crucial for protein protein interactions Although the overall structure is similar to the previously ... More
The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7–70) at 2.50 Å resolution, revealing molecular details crucial for protein–protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30’s and 40’s loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5–11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40’s loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine’s function and inhibitor development. Less
The nonpolymorphic class Ib molecule HLA-E primarily presents peptides from HLA class Ia leader peptides providing an inhibitory signal to NK cells via CD NKG interactions Although peptides of pathogenic origin can also be presented by HLA-E to T cells the molecular basis underpinning their role in antigen surveillance is largely unknown Here we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide pHLA-E from bacterial Mycobacterium tuberculosis origin and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral HIV origin The structures provided a molecular foundation to develop a novel method to introduce ... More
The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions.
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Retroviral elements from endogenous retroviruses have functions in mammalian physiology The best-known examples are the envelope proteins that function in placenta development and immune suppression Porcine endogenous retroviruses PERVs are an understudied class of endogenous retroviruses that infect cultured human cells raising concern regarding porcine xenografts The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development Despite the growing interest in PERVs their envelope glycoproteins remain poorly characterized Here we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain The PERV fusion protein structure reveals a conserved class ... More
Retroviral elements from endogenous retroviruses have functions in mammalian physiology. The best-known examples are the envelope proteins that function in placenta development and immune suppression. Porcine endogenous retroviruses (PERVs) are an understudied class of endogenous retroviruses that infect cultured human cells, raising concern regarding porcine xenografts. The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development. Despite the growing interest in PERVs, their envelope glycoproteins remain poorly characterized. Here, we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain. The PERV fusion protein structure reveals a conserved class I viral fusion protein six-helix bundle. Biophysical experiments demonstrated that the thermodynamic stability of the PERV fusion protein secondary structure was the same at physiological and acidic pHs. A conserved surface analysis highlights the high degree of sequence conservation among retroviral fusogens in the chain reversal region that facilitates the large-scale conformational change required for membrane fusion. Further structural alignment of class I viral fusogens revealed a phylogenetic clustering that shows evolution into various lineages that correlate with virus mechanisms of cell entry. Our work indicates that structural dendrograms can be used to qualitatively infer insights into the fusion mechanisms of newly discovered class I viral fusogen structures. Less
-adrenergic receptors ARs are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems The therapeutic potential of ARs however is largely unexploited and hampered by the scarcity of subtype-selective ligands Moreover several aminergic drugs either show off-target binding to ARs or fail to interact with the desired subtype Here we report the crystal structure of human BAR bound to the inverse agonist -cyclazosin enabled by the fusion to a DARPin crystallization chaperone The BAR structure allows the identification of two unique secondary binding pockets By structural comparison of BAR with ARs and by constructing BAR- CAR ... More
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype. Less
Activity-regulated cytoskeleton-associated protein Arc is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals However the mechanistic and structural basis of Arc function is little understood Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands In addition the NTD and CTD both function in Arc oligomerization including assembly of retrovirus-like capsid involved in intercellular signaling We produced and characterised six ultra-high-affinity anti-Arc nanobodies Nb The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs ... More
Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo. Less
Membrane proteins are central to many pathophysiological processes yet remain very difficult to analyze structurally Moreover high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation Here we present a facile and versatile platform for in meso membrane protein crystallization enabling rapid atomic structure determination at both cryogenic and room temperatures We apply this approach to human integral membrane proteins which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR integral membrane protein Finally we demonstrate an automated pipeline ... More
Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins. Less
Target-based azole resistance in Candida albicans involves overexpression of the ERG gene encoding lanosterol -demethylase LDM and or the presence of single or multiple mutations in this enzyme Overexpression of Candida albicans LDM CaLDM Y H I T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole and weakly increased resistance to the longer-tailed azoles VT- itraconazole and posaconazole We have used as surrogates structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM His ScLDM His to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C albicans Darlington strain ... More
Target-based azole resistance in Candida albicans involves overexpression of the ERG11 gene encoding lanosterol 14α-demethylase (LDM), and/or the presence of single or multiple mutations in this enzyme. Overexpression of Candida albicans LDM (CaLDM) Y132H I471T by the Darlington strain strongly increased resistance to the short-tailed azoles fluconazole and voriconazole, and weakly increased resistance to the longer-tailed azoles VT-1161, itraconazole and posaconazole. We have used, as surrogates, structurally aligned mutations in recombinant hexahistidine-tagged full-length Saccharomyces cerevisiae LDM6×His (ScLDM6×His) to elucidate how differential susceptibility to azole drugs is conferred by LDM of the C. albicans Darlington strain. The mutations Y140H and I471T were introduced, either alone or in combination, into ScLDM6×His via overexpression of the recombinant enzyme from the PDR5 locus of an azole hypersensitive strain of S. cerevisiae. Phenotypes and high-resolution X-ray crystal structures were determined for the surrogate enzymes in complex with representative short-tailed (voriconazole) and long-tailed (itraconazole) triazoles. The preferential high-level resistance to short-tailed azoles conferred by the ScLDM Y140H I471T mutant required both mutations, despite the I471T mutation conferring only a slight increase in resistance. Crystal structures did not detect changes in the position/tilt of the heme co-factor of wild-type ScLDM, I471T and Y140H single mutants, or the Y140H I471T double-mutant. The mutant threonine sidechain in the Darlington strain CaLDM perturbs the environment of the neighboring C-helix, affects the electronic environment of the heme, and may, via differences in closure of the neck of the substrate entry channel, increase preferential competition between lanosterol and short-tailed azole drugs. Less
Heat-shock proteins of kDa Hsp s are vital for all life and are notably important in protein folding Hsp s use ATP binding and hydrolysis at a nucleotide-binding domain NBD to control the binding and release of client polypeptides at a substrate-binding domain SBD however the mechanistic basis for this allostery has been elusive Here we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp DnaK We then develop a theoretical model for allosteric equilibria among Hsp conformational states to explain the observations a restraining state Hsp R-ATP restricts ATP hydrolysis and binds peptides poorly whereas a stimulating ... More
Heat-shock proteins of 70 kDa (Hsp70s) are vital for all life and are notably important in protein folding. Hsp70s use ATP binding and hydrolysis at a nucleotide-binding domain (NBD) to control the binding and release of client polypeptides at a substrate-binding domain (SBD); however, the mechanistic basis for this allostery has been elusive. Here, we first characterize biochemical properties of selected domain-interface mutants in bacterial Hsp70 DnaK. We then develop a theoretical model for allosteric equilibria among Hsp70 conformational states to explain the observations: a restraining state, Hsp70R-ATP, restricts ATP hydrolysis and binds peptides poorly, whereas a stimulating state, Hsp70S-ATP, hydrolyzes ATP rapidly and has high intrinsic substrate affinity but rapid binding kinetics. We support this model for allosteric regulation with DnaK structures obtained in the postulated stimulating state S with biochemical tests of the S-state interface and with improved peptide-binding-site definition in an R-state structure. Less
Henipaviruses are BSL- zoonotic pathogens responsible in humans for severe encephalitis Their V protein is a key player in the evasion of the host innate immune response We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain NTD and a -enriched C-terminal domain CTD The CTD is critical for V binding to DDB which is a cellular protein that is a component of the ubiquitin ligase E complex as well as binding to MDA and LGP which are two host sensors of viral RNA Here we serendipitously discovered that the Hendra virus V protein ... More
Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). The CTD is critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200–310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins. Less
CD is the only -transmembrane -TM spanning receptor of the immune system Its extracellular domain ECD is a cell surface marker of self that binds SIRP and inhibits macrophage phagocytosis and cancer immuno-therapy approaches in clinical trials are focused on blocking CD SIRP interaction We present the crystal structure of full length CD bound to the function-blocking antibody B H CD ECD is tethered to the TM domain via a six-residue peptide linker RVVSWF that forms an extended loop SWF loop with the fundamental role of inserting the side chains of W and F into the core of CD extracellular ... More
CD47 is the only 5-transmembrane (5-TM) spanning receptor of the immune system. Its extracellular domain (ECD) is a cell surface marker of self that binds SIRPα and inhibits macrophage phagocytosis, and cancer immuno-therapy approaches in clinical trials are focused on blocking CD47/SIRPα interaction. We present the crystal structure of full length CD47 bound to the function-blocking antibody B6H12. CD47 ECD is tethered to the TM domain via a six-residue peptide linker (114RVVSWF119) that forms an extended loop (SWF loop), with the fundamental role of inserting the side chains of W118 and F119 into the core of CD47 extracellular loop region (ECLR). Using hydrogen-deuterium exchange and molecular dynamics simulations we show that CD47’s ECLR architecture, comprised of two extracellular loops and the SWF loop, creates a molecular environment stabilizing the ECD for presentation on the cell surface. These findings provide insights into CD47 immune recognition, signaling and therapeutic intervention. Less
Respiratory syncytial virus RSV is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood and vaccination against RSV remains a public health priority The RSV F glycoprotein is a major target of neutralizing antibodies and the prefusion stabilized form of F DS-Cav is under investigation as a vaccine antigen AM is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F PreF which spans two F protomers The quality of recognizing a trimer-specific epitope makes AM valuable for probing PreF-based immunogen conformation and ... More
Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infections resulting in medical intervention and hospitalizations during infancy and early childhood, and vaccination against RSV remains a public health priority. The RSV F glycoprotein is a major target of neutralizing antibodies, and the prefusion stabilized form of F (DS-Cav1) is under investigation as a vaccine antigen. AM14 is a human monoclonal antibody with the exclusive capacity of binding an epitope on prefusion F (PreF), which spans two F protomers. The quality of recognizing a trimer-specific epitope makes AM14 valuable for probing PreF-based immunogen conformation and functionality during vaccine production. Currently, only a low-resolution (5.5 Å) X-ray structure is available of the PreF-AM14 complex, revealing few reliable details of the interface. Here, we perform complementary structural studies using X-ray crystallography and cryo-electron microscopy (cryo-EM) to provide improved resolution structures at 3.6 Å and 3.4 Å resolutions, respectively. Both X-ray and cryo-EM structures provide clear side-chain densities, which allow for accurate mapping of the AM14 epitope on DS-Cav1. The structures help rationalize the molecular basis for AM14 loss of binding to RSV F monoclonal antibody-resistant mutants and reveal flexibility for the side chain of a key antigenic residue on PreF. This work provides the basis for a comprehensive understanding of RSV F trimer specificity with implications in vaccine design and quality assessment of PreF-based immunogens. Less
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process However the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood Here integrated enzymatic assays protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel B cellulase from Bacillus licheniformis Hydrolytic efficiency substrate binding affinity cleavage patterns and the apparent processivity of bacterial BlCel B are significantly impacted by the cellulose size and its surface morphology BlCel B crystallographic structure was solved with ligands spanning - to - and to subsites ... More
Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis. Hydrolytic efficiency, substrate binding affinity, cleavage patterns, and the apparent processivity of bacterial BlCel48B are significantly impacted by the cellulose size and its surface morphology. BlCel48B crystallographic structure was solved with ligands spanning -5 to -2 and +1 to +2 subsites. Statistical coupling analysis and molecular dynamics show that co-evolved residues on active site are critical for stabilizing ligands in the catalytic tunnel. Our results provide mechanistic insights into BlCel48B molecular-level determinants of activity, substrate binding, and processivity on insoluble cellulose, thus shedding light on structure-activity correlations of GH48 family members in general. Less
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus Here we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs IgAs and IgGs reactive with human norovirus HuNoV genogroup I or II GI or GII We note three binding patterns and identify monoclonal antibodies mAbs that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen HBGA blocking assay The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO- ... More
The rational development of norovirus vaccine candidates requires a deep understanding of the antigenic diversity and mechanisms of neutralization of the virus. Here, we isolate and characterize a panel of broadly cross-reactive naturally occurring human monoclonal IgMs, IgAs and IgGs reactive with human norovirus (HuNoV) genogroup I or II (GI or GII). We note three binding patterns and identify monoclonal antibodies (mAbs) that neutralize at least one GI or GII HuNoV strain when using a histo-blood group antigen (HBGA) blocking assay. The HBGA blocking assay and a virus neutralization assay using human intestinal enteroids reveal that the GII-specific mAb NORO-320, mediates HBGA blocking and neutralization of multiple GII genotypes. The Fab form of NORO-320 neutralizes GII.4 infection more potently than the mAb, however, does not block HBGA binding. The crystal structure of NORO-320 Fab in complex with GII.4 P-domain shows that the antibody recognizes a highly conserved region in the P-domain distant from the HBGA binding site. Dynamic light scattering analysis of GII.4 virus-like particles with mAb NORO-320 shows severe aggregation, suggesting neutralization is by steric hindrance caused by multivalent cross-linking. Aggregation was not observed with the Fab form of NORO-320, suggesting that this clone also has additional inhibitory features. Less
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase Lit Lit creates a lipoprotein that is less immunogenic possibly enabling the bacteria to gain a foothold in the host by stealth Here we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action Lit consists of four transmembrane helices with an extracellular cap Conserved residues map to ... More
Lipoproteins serve diverse functions in the bacterial cell and some are essential for survival. Some lipoproteins are adjuvants eliciting responses from the innate immune system of the host. The growing list of membrane enzymes responsible for lipoprotein synthesis includes the recently discovered lipoprotein intramolecular transacylase, Lit. Lit creates a lipoprotein that is less immunogenic, possibly enabling the bacteria to gain a foothold in the host by stealth. Here, we report the crystal structure of the Lit enzyme from Bacillus cereus and describe its mechanism of action. Lit consists of four transmembrane helices with an extracellular cap. Conserved residues map to the cap-membrane interface. They include two catalytic histidines that function to effect unimolecular transacylation. The reaction involves acyl transfer from the sn-2 position of the glyceryl moiety to the amino group on the N-terminal cysteine of the substrate via an 8-membered ring intermediate. Transacylation takes place in a confined aromatic residue-rich environment that likely evolved to bring distant moieties on the substrate into proximity and proper orientation for catalysis. Less
The chemokine receptor CCR plays a vital role in immune surveillance and inflammation However molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive Here we report three cryo-electron microscopy structures of Gi protein-coupled CCR in a ligand-free state and in complex with the chemokine MIP- or RANTES as well as the crystal structure of MIP- -bound CCR These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR Together with functional data the structures demonstrate that chemokine-induced rearrangement of toggle switch and ... More
The chemokine receptor CCR5 plays a vital role in immune surveillance and inflammation. However, molecular details that govern its endogenous chemokine recognition and receptor activation remain elusive. Here we report three cryo-electron microscopy structures of Gi1 protein-coupled CCR5 in a ligand-free state and in complex with the chemokine MIP-1α or RANTES, as well as the crystal structure of MIP-1α-bound CCR5. These structures reveal distinct binding modes of the two chemokines and a specific accommodate pattern of the chemokine for the distal N terminus of CCR5. Together with functional data, the structures demonstrate that chemokine-induced rearrangement of toggle switch and plasticity of the receptor extracellular region are critical for receptor activation, while a conserved tryptophan residue in helix II acts as a trigger of receptor constitutive activation. Less
Odorant-binding proteins OBPs as they occur in insects form a distinct class of proteins that apparently has no closely related representatives in other animals However ticks mites spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs In this work we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor a major pest of honey bee Varroa OBPs present six cysteines paired into three disulphide bridges but with positions in the sequence and connections different from those of their insect counterparts VdesOBP structure was determined in two closely related crystal forms ... More
Odorant-binding proteins (OBPs), as they occur in insects, form a distinct class of proteins that apparently has no closely related representatives in other animals. However, ticks, mites, spiders and millipedes contain genes encoding proteins with sequence similarity to insect OBPs. In this work, we have explored the structure and function of such non-insect OBPs in the mite Varroa destructor, a major pest of honey bee. Varroa OBPs present six cysteines paired into three disulphide bridges, but with positions in the sequence and connections different from those of their insect counterparts. VdesOBP1 structure was determined in two closely related crystal forms and appears to be a monomer. Its structure assembles five α-helices linked by three disulphide bridges, one of them exhibiting a different connection as compared to their insect counterparts. Comparison with classical OBPs reveals that the second of the six α-helices is lacking in VdesOBP1. Ligand-binding experiments revealed molecules able to bind only specific OBPs with a moderate affinity, suggesting that either optimal ligands have still to be identified, or post-translational modifications present in the native proteins may be essential for modulating binding activity, or else these OBPs might represent a failed attempt in evolution and are not used by the mites. Less
Plant pathogens cause disease through secreted effector proteins which act to modulate host physiology and promote infection Typically the sequences of effectors provide little functional information and further targeted experimentation is required Here we utilised a structure function approach to study SnTox an effector from the necrotrophic fungal pathogen Parastagonospora nodorum which causes cell death in wheat-lines carrying the sensitivity gene Snn We developed a workflow for the production of SnTox in a heterologous host that enabled crystal structure determination We show this approach can be successfully applied to effectors from other pathogenic fungi Complementing this an in-silico study uncovered ... More
Plant pathogens cause disease through secreted effector proteins, which act to modulate host physiology and promote infection. Typically, the sequences of effectors provide little functional information and further targeted experimentation is required. Here, we utilised a structure/function approach to study SnTox3, an effector from the necrotrophic fungal pathogen Parastagonospora nodorum, which causes cell death in wheat-lines carrying the sensitivity gene Snn3. We developed a workflow for the production of SnTox3 in a heterologous host that enabled crystal structure determination. We show this approach can be successfully applied to effectors from other pathogenic fungi. Complementing this, an in-silico study uncovered the prevalence of an expanded subclass of effectors from fungi. The β-barrel fold of SnTox3 is a novel fold among fungal effectors. We demonstrate that SnTox3 is a pre-pro-protein and that the protease Kex2 removes the pro-domain. Our in-silico studies suggest that Kex2-processed pro-domain (designated here as K2PP) effectors are common in fungi, and we demonstrate this experimentally for effectors from Fusarium oxysporum f sp. lycopersici. We propose that K2PP effectors are highly prevalent among fungal effectors. The identification and classification of K2PP effectors has broad implications for the approaches used to study their function in fungal virulence. Less
The design of peptides that assemble in membranes to form functional ion channels is challenging Specifically hydrophobic interactions must be designed between the peptides and at the peptide lipid interfaces simultaneously Here we take a multi-step approach towards this problem First we use rational de novo design to generate water-soluble -helical barrels with polar interiors and confirm their structures using high-resolution X-ray crystallography These -helical barrels have water-filled lumens like those of transmembrane channels Next we modify the sequences to facilitate their insertion into lipid bilayers Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse cation-selective ... More
The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide–lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Next, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from the lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide. Less
An effective HIV- vaccine will likely need to elicit broadly neutralizing antibodies bNAbs Broad and potent VRC -class bNAbs have been isolated from multiple infected individuals suggesting that they could be reproducibly elicited by vaccination Several HIV- envelope-derived germline-targeting immunogens have been designed to engage naive VRC -class precursor B cells However they also present off-target epitopes that could hinder development of VRC -class bNAbs We characterize a panel of anti-idiotypic monoclonal antibodies ai-mAbs raised against inferred-germline iGL VRC -class antibodies By leveraging binding structural and B cell sorting data we engineered a bispecific molecule derived from two ai-mAbs one ... More
An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo. Less
Antibodies are crucial to immune protection against SARS-CoV- with some in emergency use as therapeutics Here we identify human monoclonal antibodies mAbs recognizing the virus spike and focus mainly on that bind the receptor binding domain RBD We devise a competition data-driven method to map RBD binding sites We find that although antibody binding sites are widely dispersed neutralizing antibody binding is focused with nearly all highly inhibitory mAbs IC mg mL blocking receptor interaction except for one that binds a unique epitope in the N-terminal domain Many of these neutralizing mAbs use public Vgenes and are close to germline ... More
Antibodies are crucial to immune protection against SARS-CoV-2, with some in emergency use as therapeutics. Here, we identify 377 human monoclonal antibodies (mAbs) recognizing the virus spike and focus mainly on 80 that bind the receptor binding domain (RBD). We devise a competition data-driven method to map RBD binding sites. We find that although antibody binding sites are widely dispersed, neutralizing antibody binding is focused, with nearly all highly inhibitory mAbs (IC50 < 0.1 mg/mL) blocking receptor interaction, except for one that binds a unique epitope in the N-terminal domain. Many of these neutralizing mAbs use public Vgenes and are close to germline. We dissect the structural basis of recognition for this large panel of antibodies through X-ray crystallography and cryoelectron microscopy of 19 Fab-antigen structures. We find novel binding modes for some potently inhibitory antibodies and demonstrate that strongly neutralizing mAbs protect, prophylactically or therapeutically, in animal models. Less
A bispecific antibody BsAb targeting the epidermal growth factor receptor EGFR and mesenchymal epithelial transition factor MET pathways represents a novel approach to overcome resistance to targeted therapies in patients with non small cell lung cancer In this study we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule The BsAbs were derived from different EGFR and MET parental monoclonal antibodies Initially molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET Hits were identified and further screened based on their potential to induce ... More
A bispecific antibody (BsAb) targeting the epidermal growth factor receptor (EGFR) and mesenchymal–epithelial transition factor (MET) pathways represents a novel approach to overcome resistance to targeted therapies in patients with non–small cell lung cancer. In this study, we sequentially screened a panel of BsAbs in a combinatorial approach to select the optimal bispecific molecule. The BsAbs were derived from different EGFR and MET parental monoclonal antibodies. Initially, molecules were screened for EGFR and MET binding on tumor cell lines and lack of agonistic activity toward MET. Hits were identified and further screened based on their potential to induce untoward cell proliferation and cross-phosphorylation of EGFR by MET via receptor colocalization in the absence of ligand. After the final step, we selected the EGFR and MET arms for the lead BsAb and added low fucose Fc engineering to generate amivantamab (JNJ-61186372). The crystal structure of the anti-MET Fab of amivantamab bound to MET was solved, and the interaction between the two molecules in atomic details was elucidated. Amivantamab antagonized the hepatocyte growth factor (HGF)-induced signaling by binding to MET Sema domain and thereby blocking HGF β-chain—Sema engagement. The amivantamab EGFR epitope was mapped to EGFR domain III and residues K443, K465, I467, and S468. Furthermore, amivantamab showed superior antitumor activity over small molecule EGFR and MET inhibitors in the HCC827-HGF in vivo model. Based on its unique mode of action, amivantamab may provide benefit to patients with malignancies associated with aberrant EGFR and MET signaling. Less
Nicotinamide adenine dinucleotide NAD is a key molecule in cellular bioenergetics and signalling Various bacterial pathogens release NADase enzymes into the host cell that deplete the host s NAD pool thereby causing rapid cell death Here we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa The enzymes harbour a tuberculosis necrotizing toxin TNT domain and are predominately present in pathogenic species The X-ray structure of the homodimeric A fumigatus protein reveals unique properties including N-linked glycosylation and a Ca -binding site whose occupancy regulates activity The structure ... More
Nicotinamide adenine dinucleotide (NAD) is a key molecule in cellular bioenergetics and signalling. Various bacterial pathogens release NADase enzymes into the host cell that deplete the host’s NAD+ pool, thereby causing rapid cell death. Here, we report the identification of NADases on the surface of fungi such as the pathogen Aspergillus fumigatus and the saprophyte Neurospora crassa. The enzymes harbour a tuberculosis necrotizing toxin (TNT) domain and are predominately present in pathogenic species. The 1.6 Å X-ray structure of the homodimeric A. fumigatus protein reveals unique properties including N-linked glycosylation and a Ca2+-binding site whose occupancy regulates activity. The structure in complex with a substrate analogue suggests a catalytic mechanism that is distinct from those of known NADases, ADP-ribosyl cyclases and transferases. We propose that fungal NADases may convey advantages during interaction with the host or competing microorganisms. Less
Aiming at streamlining GPCR production from E coli inclusion bodies for structural analysis we present a generic approach to assess and optimize refolding yield through thermostability analysis Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent -Diethylamino- - -maleimidophenyl - -methylcoumarin CPM Successful expression purification and refolding is shown for two G protein-coupled receptors GPCR the sphingosine- -phosphate receptor S P and the orphan receptor GPR Refolded receptors were subjected to lipidic cubic phase crystallization screening
The human neuropeptide Y NPY Y receptor Y R plays essential roles in food intake bone formation and mood regulation and has been considered an important drug target for obesity and anxiety However development of drugs targeting Y R remains challenging with no success in clinical application yet Here we report the crystal structure of Y R bound to a selective antagonist JNJ- at resolution The structure reveals molecular details of the ligand-binding mode of Y R Combined with mutagenesis studies the Y R structure provides insights into key factors that define antagonistic activity of diverse antagonists Comparison with the ... More
The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank. Less
Myxococcus xanthus displays two types of motilities i e Social S and Adventurous A The pole-to-pole reversals of these motility regulator proteins is the key to this process Here we determined resolution crystal structure of MglC which revealed that despite sharing sequence identity both MglB and MglC adopt Regulatory Light Chain RLC family fold Interestingly MglC is structurally unique compared to the other known RLC family proteins having - shift in the orientation of functionally important helix Using isothermal titration calorimetry and gel filtration chromatography we show that MglC binds MglB in stoichiometry with submicromolar range dissociation constant Using combination ... More
Myxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 Å resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30°-40° shift in the orientation of functionally important α2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB. Less
Neurotensin receptor NTSR and related G protein coupled receptors of the ghrelin family are clinically unexploited and several mechanistic aspects of their activation and inactivation have remained unclear Enabled by a new crystallization design we present five new structures apo-state NTSR as well as complexes with nonpeptide inverse agonists SR and SR A partial agonist RTI- a and the novel full agonist SRI- providing structural rationales on how ligands modulate NTSR The inverse agonists favor a large extracellular opening of helices VI and VII undescribed so far for NTSR causing a constriction of the intracellular portion In contrast the full ... More
Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors. Less
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase LCP However almost all previous crystallization trials have used monoacylglycerols with - cis- -octadecanoyl -rac-glycerol MO being the most widely used lipid We now report that EROCOC mixed with w w cholesterol Fig serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A A receptor A AR The structures of EROCOC -matrix grown A AR crystals were determined at resolution by serial synchrotron rotation crystallography at a cryogenic temperature and at ... More
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein and substrate engineering based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F A MD simulation further ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia CPVT In vivo calsequestrin multimerizes into filaments but a compelling atomic-resolution structure of a calsequestrin filament is lacking We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay We also report and characterize a novel disease-associated calsequestrin mutation S I which localizes to the filament-forming interface In addition we show that a previously reported dominant disease mutation K R maps to the same multimerization surface Both mutations disrupt filamentation suggesting that dominant disease ... More
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay. We also report and characterize a novel disease-associated calsequestrin mutation, S173I, which localizes to the filament-forming interface. In addition, we show that a previously reported dominant disease mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation, suggesting that dominant disease arises from defects in multimer formation. A ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our understanding of calsequestrin biochemistry and provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias. Less
Fungal aryl-alcohol oxidases AAOx are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids with concomitant production of hydrogen peroxide H O as by-product The H O can be provided to lignin degradation pathway a biotechnological property explored in biofuel production In the thermophilic fungus Thermothelomyces thermophilus formerly Myceliophthora thermophila just one AAOx was identified in the exo-proteome
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates one from the main group M and the other from the minor group O of HIV- we identify in the C-terminal domain CTD of integrase a new functional motif constituted by four non-contiguous amino acids N K N K Mutating the lysines abolishes integration through decreased -processing and inefficient nuclear import of reverse transcribed genomes Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration The number of charges in the motif appears ... More
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3’-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. Less
Immuno-oncology approaches that utilize T cell receptors TCRs are becoming highly attractive because of their potential to target virtually all cellular proteins including cancer-specific epitopes via the recognition of peptide-human leukocyte antigen pHLA complexes presented at the cell surface However because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities efforts have been made to enhance their binding strength in some cases by several million-fold In this study we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors Additionally we performed molecular dynamics simulations ... More
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment. Less
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases EHs reveals its resourceful microscopic machinery The whole-genome sequence of Streptomyces sp CBMAI an endophytic actinobacterium isolated from Citrus sinensis branches was explored by genome mining and a putative -epoxide hydrolase named B EPH and encoded by amino acids was selected for functional and structural studies The crystal structure of B EPH was obtained at a resolution of and ... More
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications. Less
T cell-mediated immunity is governed primarily by T cell receptor TCR recognition of peptide-human leukocyte antigen pHLA complexes and is essential for immunosurveillance and disease control This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region CDR loops and whereas peptide selectivity is guided by direct interactions with the TCR CDR loops Here we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A MAGE-A The TCR bound pHLA in a position shifted toward the peptide's N terminus This enabled the ... More
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. Less
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational switching that underpin their gene-regulating capabilities Previous structural studies on the flavin mononucleotide FMN -binding aptamer of the FMN riboswitch however have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior We have determined a - resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures particularly in the conformation and orientation of the P and P helices The nearly symmetrical tertiary structure provides a mechanism ... More
Knowledge of both apo and holo states of riboswitches aid in elucidating the various mechanisms of ligand-induced conformational “switching” that underpin their gene-regulating capabilities. Previous structural studies on the flavin mononucleotide (FMN)-binding aptamer of the FMN riboswitch, however, have revealed minimal conformational changes associated with ligand binding that do not adequately explain the basis for the switching behavior. We have determined a 2.7-Å resolution crystal structure of the ligand-free FMN riboswitch aptamer that is distinct from previously reported structures, particularly in the conformation and orientation of the P1 and P4 helices. The nearly symmetrical tertiary structure provides a mechanism by which one of two pairs of adjacent helices (P3/P4 or P1/P6) undergo collinear stacking in a mutually exclusive manner, in the absence or presence of ligand, respectively. Comparison of these structures suggests the stem-loop that includes P4 and L4 is important for maintaining a global conformational state that, in the absence of ligand, disfavors formation of the P1 regulatory helix. Together, these results provide further insight to the structural basis for conformational switching of the FMN riboswitch. Less
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi Trypanosoma brucei and Leishmania major Tritryp published in opened up new perspectives for drug development targeting Chagas disease African sleeping sickness and Leishmaniasis neglected diseases affecting millions of most economically disadvantaged people Still half of the Tritryp genes code for proteins of unknown function Moreover almost of conserved eukaryotic protein domains are missing in the Tritryp genomes This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes Furthermore proteins without homologous counterparts in humans may provide ... More
Complete genome sequencing of the kinetoplastid protozoans Trypanosoma cruzi, Trypanosoma brucei and Leishmania major (Tritryp), published in 2005, opened up new perspectives for drug development targeting Chagas disease, African sleeping sickness and Leishmaniasis, neglected diseases affecting millions of most economically disadvantaged people. Still, half of the Tritryp genes code for proteins of unknown function. Moreover, almost 50% of conserved eukaryotic protein domains are missing in the Tritryp genomes. This suggests that functional and structural characterization of proteins of unknown function could reveal novel protein folds used by the trypanosomes for common cellular processes. Furthermore, proteins without homologous counterparts in humans may provide potential targets for therapeutic intervention. Here we describe the crystal structure of the T. cruzi protein Q4D6Q6, a conserved and kinetoplastid-specific protein essential for cell viability. Q4D6Q6 is a representative of a family of 20 orthologs, all annotated as proteins of unknown function. Q4D6Q6 monomers adopt a ββαββαββ topology and form a propeller-like tetramer. Oligomerization was verified in solution using NMR, SAXS, analytical ultra-centrifugation and gel filtration chromatography. A rigorous search for similar structures using the DALI server revealed similarities with propeller-like structures of several different functions. Although a Q4D6Q6 function could not be inferred from such structural comparisons, the presence of an oxidized cysteine at position 69, part of a cluster with phosphorylated serines and hydrophobic residues, identifies a highly reactive site and suggests a role of this cysteine as a nucleophile in a post-translational modification reaction. Less
NKp is one of the main human natural killer NK cell activating receptors used in directed immunotherapy The oligomerization of the NKp ligand binding domain depends on the length of the C-terminal stalk region but our structural knowledge of NKp oligomerization and its role in signal transduction remains limited Moreover ligand binding of NKp is affected by the presence and type of N-glycosylation In this study we assessed whether NKp oligomerization depends on its N-glycosylation Our results show that NKp forms oligomers when expressed in HEK S GnTI cell lines with simple N-glycans However NKp was detected only as monomers ... More
NKp30 is one of the main human natural killer (NK) cell activating receptors used in directed immunotherapy. The oligomerization of the NKp30 ligand binding domain depends on the length of the C-terminal stalk region, but our structural knowledge of NKp30 oligomerization and its role in signal transduction remains limited. Moreover, ligand binding of NKp30 is affected by the presence and type of N-glycosylation. In this study, we assessed whether NKp30 oligomerization depends on its N-glycosylation. Our results show that NKp30 forms oligomers when expressed in HEK293S GnTI− cell lines with simple N-glycans. However, NKp30 was detected only as monomers after enzymatic deglycosylation. Furthermore, we characterized the interaction between NKp30 and its best-studied cognate ligand, B7-H6, with respect to glycosylation and oligomerization, and we solved the crystal structure of this complex with glycosylated NKp30, revealing a new glycosylation-induced mode of NKp30 dimerization. Overall, this study provides new insights into the structural basis of NKp30 oligomerization and explains how the stalk region and glycosylation of NKp30 affect its ligand affinity. This furthers our understanding of the molecular mechanisms involved in NK cell activation, which is crucial for the successful design of novel NK cell-based targeted immunotherapeutics. Less
T cell recognition of peptides presented by human leukocyte antigens HLAs is mediated by the highly variable T cell receptor TCR Despite this built-in TCR variability individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD T cells Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA However few examples exist for CD T cells Here we investigate CD T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity We identify five internal epitopes that are ... More
T cell recognition of peptides presented by human leukocyte antigens (HLAs) is mediated by the highly variable T cell receptor (TCR). Despite this built-in TCR variability, individuals can mount immune responses against viral epitopes by using identical or highly related TCRs expressed on CD8+ T cells. Characterization of these TCRs has extended our understanding of the molecular mechanisms that govern the recognition of peptide-HLA. However, few examples exist for CD4+ T cells. Here, we investigate CD4+ T cell responses to the internal proteins of the influenza A virus that correlate with protective immunity. We identify five internal epitopes that are commonly recognized by CD4+ T cells in five HLA-DR1+ subjects and show conservation across viral strains and zoonotic reservoirs. TCR repertoire analysis demonstrates several shared gene usage biases underpinned by complementary biochemical features evident in a structural comparison. These epitopes are attractive targets for vaccination and other T cell therapies. Less
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses Viral replication requires a C-like cysteine protease CLpro which processes the kDa viral polyprotein into six functional proteins The CLpro has attracted much interest due to its potential as a target for antiviral drugs A system for growing high-quality crystals of native Southampton norovirus CLpro SV CP has been established allowing the ligand-free crystal structure to be determined to in a tetrameric state This also allowed crystal-based fragment screening to be performed with various compound libraries ultimately to guide drug discovery for SV CP A total of fragments were ... More
Outbreaks of human epidemic nonbacterial gastroenteritis are mainly caused by noroviruses. Viral replication requires a 3C-like cysteine protease (3CLpro) which processes the 200 kDa viral polyprotein into six functional proteins. The 3CLpro has attracted much interest due to its potential as a target for antiviral drugs. A system for growing high-quality crystals of native Southampton norovirus 3CLpro (SV3CP) has been established, allowing the ligand-free crystal structure to be determined to 1.3 Å in a tetrameric state. This also allowed crystal-based fragment screening to be performed with various compound libraries, ultimately to guide drug discovery for SV3CP. A total of 19 fragments were found to bind to the protease out of the 844 which were screened. Two of the hits were located at the active site of SV3CP and showed good inhibitory activity in kinetic assays. Another 5 were found at the enzyme’s putative RNA-binding site and a further 11 were located in the symmetric central cavity of the tetramer. Less
Ketol-acid reductoisomerase KARI the second enzyme in the branched-chain amino acid biosynthesis pathway is a potential drug target for bacterial infections including Mycobacterium tuberculosis Here we have screened the Medicines for Malaria Venture Pathogen Box against purified M tuberculosis Mt KARI and identified two compounds that have Ki values below nm In Mt cell susceptibility assays one of these compounds exhibited an IC value of m Co-crystallization of this compound - methylsulfonyl methyl - H-benzo b oxazin- -one MMV in complex with Staphylococcus aureus KARI which has identity with Mt KARI NADPH and Mg yielded a structure to resolution However ... More
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have Ki values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC50 value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg2+ yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data. Less
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing Co-crystallization with proteins is a straight forward approach to overcome these challenges The human RNA-binding protein U A has previously been established as crystallization module however the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U A system Here we report three crystal structures of tryptophan-containing U A variants which expand the crystallization toolbox for nucleic acids Analysis of the structures complemented by SAXS NMR spectroscopy and optical spectroscopy allow for insights into the potential of the U ... More
The major bottlenecks in structure elucidation of nucleic acids are crystallization and phasing. Co-crystallization with proteins is a straight forward approach to overcome these challenges. The human RNA-binding protein U1A has previously been established as crystallization module, however, the absence of UV-active residues and the predetermined architecture in the asymmetric unit constitute clear limitations of the U1A system. Here, we report three crystal structures of tryptophan-containing U1A variants, which expand the crystallization toolbox for nucleic acids. Analysis of the structures complemented by SAXS, NMR spectroscopy, and optical spectroscopy allow for insights into the potential of the U1A variants to serve as crystallization modules for nucleic acids. In addition, we report a fast and efficient protocol for crystallization of RNA by soaking and present a fluorescence-based approach for detecting RNA-binding in crystallo. Our results provide a new tool set for the crystallization of RNA and RNA:DNA complexes. Less
Tumor-associated peptide human leukocyte antigen complexes pHLAs represent the largest pool of cell surface expressed cancer-specific epitopes making them attractive targets for cancer therapies Soluble bispecific molecules that incorporate an anti-CD effector function are being developed to redirect T cells against these targets using different approaches The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs e g immune-mobilizing monoclonal T cell receptors against cancer ImmTAC molecules whereas the second harnesses an antibody-based format TCR-mimic antibodies For both classes of reagent target specificity is vital considering the vast universe of potential pHLA molecules that can be presented on healthy ... More
Tumor-associated peptide–human leukocyte antigen complexes (pHLAs) represent the largest pool of cell surface–expressed cancer-specific epitopes, making them attractive targets for cancer therapies. Soluble bispecific molecules that incorporate an anti-CD3 effector function are being developed to redirect T cells against these targets using 2 different approaches. The first achieves pHLA recognition via affinity-enhanced versions of natural TCRs (e.g., immune-mobilizing monoclonal T cell receptors against cancer [ImmTAC] molecules), whereas the second harnesses an antibody-based format (TCR-mimic antibodies). For both classes of reagent, target specificity is vital, considering the vast universe of potential pHLA molecules that can be presented on healthy cells. Here, we made use of structural, biochemical, and computational approaches to investigate the molecular rules underpinning the reactivity patterns of pHLA-targeting bispecifics. We demonstrate that affinity-enhanced TCRs engage pHLA using a comparatively broad and balanced energetic footprint, with interactions distributed over several HLA and peptide side chains. As ImmTAC molecules, these TCRs also retained a greater degree of pHLA selectivity, with less off-target activity in cellular assays. Conversely, TCR-mimic antibodies tended to exhibit binding modes focused more toward hot spots on the HLA surface and exhibited a greater degree of crossreactivity. Our findings extend our understanding of the basic principles that underpin pHLA selectivity and exemplify a number of molecular approaches that can be used to probe the specificity of pHLA-targeting molecules, aiding the development of future reagents. Less