901 Citations
Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein Dpr as a defensive mechanism against oxidative stress Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle Dpr as other members of the family uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers N-terminal pores to take up Fe and deposit it inside the cavity after its oxidation to Fe in ferroxidase sites in the interior of the dodecamer In this way the generation of toxic hydroxyl radicals ... More
Streptococcus suis is an emerging catalase-negative zoonotic pathogen that uses the peroxide resistance protein (Dpr) as a defensive mechanism against oxidative stress. Dpr belongs to a family of proteins that form spherical dodecamers with a hollow cavity in the middle. Dpr, as other members of the family, uses four pores found on the surface of the dodecamer and formed by the N-terminals of adjacent monomers (N-terminal pores) to take up Fe2+ and deposit it inside the cavity after its oxidation to Fe3+ in ferroxidase sites in the interior of the dodecamer. In this way, the generation of toxic hydroxyl radicals via Fenton’s reaction is avoided. In this study, a new purification process and crystallization conditions for Dpr were found. Besides, the ligandability of Dpr for use as a drug target was investigated. 6xHis-tagged Dpr was successfully produced and purified. Crystallization screens yielded crystals in 10 conditions and further optimization led to crystals suitable for structural analysis. Synchrotron X-ray data were collected to 2.2 Å resolution. A novel ligand library design led to an initial library of 82 compounds that could act as possible N-terminal pore blockers. After a score threshold of -7, twenty (20) ligands remained. Similar, to the latter ones, marketed ligands were retrieved, and ten (10) of them were kept, all sharing the feature of having aromatic rings. Phe133 was found as the only residue responsible for Pi-pi interactions with the ligands. This is the first successful approach for 6xHis-tag Dpr crystal production and structure determination. It is also the first approach for ligand creation against the N-terminal pores of the Dpr, setting the basis for new possible future therapeutic approaches for S. suis-related infections treatment, avoiding the obstacle of antibiotic resistance. Less
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation recent advances in methods for data collection from multiple crystals have overcome this issue At SPring- an in situ diffraction measurement system was constructed consisting of a goniometer for a plate an articulated robot and plate storage Using this system complete data sets were obtained utilizing the small-wedge measurement method Combining this system with an ... More
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser. Less
Viruses are responsible for some of the most deadly human diseases yet available vaccines and antivirals address only a fraction of the potential viral human pathogens Here we provide a methodology for managing human herpesvirus HHV infection by covalently inactivating the HHV maturational protease via a conserved non-catalytic cysteine C Using human cytomegalovirus protease HCMV Pr as a model we screened a library of disulfides to identify molecules that tether to C and inhibit proteolysis then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs We further developed an optimized tool compound targeted ... More
Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
High-throughput experimentation HTE seeks to accelerate the exploration of materials space by uniting robotics combinatorial methods and parallel processing HTE is particularly relevant to metal halide perovskites MHPs a diverse class of optoelectronic materials with a large chemical space Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP methoxy-phenethylammonium lead iodide -MeO-PEAI -PbI We then synthesize MHP single crystals and measure their photoluminescence ... More
High-throughput experimentation (HTE) seeks to accelerate the exploration of materials space by uniting robotics, combinatorial methods, and parallel processing. HTE is particularly relevant to metal halide perovskites (MHPs), a diverse class of optoelectronic materials with a large chemical space. Here we develop an HTE workflow to synthesize and characterize light-emitting MHP single crystals, allowing us to generate the first reported data set of experimentally derived photoluminescence spectra for low-dimensional MHPs. We leverage the accelerated workflow to optimize the synthesis and emission of a new MHP, methoxy-phenethylammonium lead iodide ((4-MeO-PEAI)2-PbI2). We then synthesize 16 000 MHP single crystals and measure their photoluminescence to study the effects of synthesis parameters and compositional engineering on the emission intensity of 54 distinct MHPs: we achieve an acceleration factor of more than 100 times over previously reported HTE MHP synthesis and characterization methods. Using insights derived from this analysis, we screen an existing database for new, potentially emissive MHPs. On the basis of the Tanimoto similarity of the bright available emitters, we present our top candidates for future exploration. As a proof of concept, we use one of these (3,4-difluorophenylmethanamine) to synthesize an MHP which we find has a photoluminescence quantum yield of 10%. Less
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
Broadly HIV- neutralizing VRC -class antibodies bind the CD -binding site of Env and contain VH - derived heavy chains paired with light chains expressing five amino acid long CDRL s Their unmutated germline forms do not recognize HIV- Env and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding na ve B cells by the vaccine immunogens To address this point we examined Env-specific B cell receptor sequences from participants in the HVTN clinical trial Of all the sequences analyzed only one displayed homology to VRC -class antibodies but ... More
Broadly HIV-1–neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02–derived heavy chains paired with light chains expressing five–amino acid–long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line–targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans. Less
Chemically induced dimerization CID is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes The induced heterodimerization of FKBP fusion proteins by rapamycin and FK has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes Here we report the crystal structure of the AtFKBP FKBD in complex with rapamycin The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD The triene arm of rapamycin appears to ... More
Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes. Less
Although monoclonal antibodies mAbs have been shown to be extremely effective in treating a number of diseases they often suffer from poor developability attributes such as high viscosity and low solubility at elevated concentrations Since experimental candidate screening is often materials and labor intensive there is substantial interest in developing in silico tools for expediting mAb design Here we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility The extrapolated protein solubilities of a set of antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay D homology models ... More
Although monoclonal antibodies (mAbs) have been shown to be extremely effective in treating a number of diseases, they often suffer from poor developability attributes, such as high viscosity and low solubility at elevated concentrations. Since experimental candidate screening is often materials and labor intensive, there is substantial interest in developing in silico tools for expediting mAb design. Here, we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility. The extrapolated protein solubilities of a set of 111 antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay. 3D homology models of the antibodies were determined, and a large set of in house and commercially available molecular descriptors were then calculated. The resulting experimental and descriptor data were then used for the development of QSAR models of mAb solubilities. After feature selection and training with different machine learning algorithms, the models were evaluated with external test sets. The resulting regression models were able to estimate the solubility values of external test set data with R2 of 0.81 and 0.85 for the two regression models developed. In addition, three class and binary classification models were developed and shown to be good estimators of mAb solubility behavior, with overall test set accuracies of 0.70 and 0.95, respectively. The analysis of the selected molecular descriptors in these models was also found to be informative and suggested that several charge-based descriptors and isotype may play important roles in mAb solubility. The combination of high throughput relative solubility experimental techniques in concert with efficient machine learning QSAR models offers an opportunity to rapidly screen potential mAb candidates and to design therapeutics with improved solubility characteristics. 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
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins MPs at a molecular level High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs two different proton pumps and a sodium light-driven ion pump The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs a surface usually accommodating hydrophobic chains of annular lipids which are known structural and functional determinants for MPs In conformity ... More
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs – a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia. Less
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past years The most current and widespread of these is SARS-CoV- The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection We previously identified a neutralizing monoclonal antibody CV - that binds to the SARS-CoV- spike neutralizes the SARS-CoV- Beta variant comparably to the ancestral Wuhan Hu- ... More
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Less
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa dubbed the green rush In transgenic synthetic biology applications NphB is a promiscuous prenyltransferase from Streptomyces sp often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid CBGA the key precursor to many other cannabinoids However its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA Herein we demonstrated a detailed and extensive computational ... More
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years. This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa, dubbed the green rush. In transgenic synthetic biology applications, NphB is a promiscuous prenyltransferase from Streptomyces sp. often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid (CBGA), the key precursor to many other cannabinoids. However, its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA. Herein, we demonstrated a detailed and extensive computational structure-guided approach in identifying target residues of mutation for engineering NphB for optimal CBGA production. Our comprehensive computational workflow has led to the discovery of several highly regiospecific variants that produce CBGA exclusively, with the best-performing V49W/Y288P variant having a 13.6-fold yield improvement, outperforming all previous work on NphB enzyme engineering. We subsequently investigated the effects of these mutations by X-ray crystallographic studies of the mutant variants and performed molecular dynamics simulations to uncover an interplay of a H-bonding network and an optimal ligand orientation that favors the CBGA production over the side product. Collectively, this study not only recapitulates the utility of computational tools in informing and accelerating experimental design but also contributes to a better understanding of molecular mechanisms that govern enzyme regioselectivity and readily aids in cannabinoid synthetic biology production for future research into maximizing their therapeutic potential. Less
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase LCP To date there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability Here we report small-angle X-ray scattering measurements of a monoolein water mixture during continuous delivery using a high viscosity injector We observe both an alignment and modification of ... More
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase (LCP). To date, there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase. This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability. Here we report small-angle X-ray scattering measurements of a monoolein:water mixture during continuous delivery using a high viscosity injector. We observe both an alignment and modification of the LCP as a direct result of the injection process. The orientation of the cubic lattice with respect to the beam was estimated based on the anisotropy of the diffraction pattern and does not correspond to a single low order zone axis. The solvent fraction was also observed to impact the stability of the cubic phase during injection. In addition, depending on the distance traveled by the lipid after exiting the needle, the phase is observed to transition from a pure diamond phase () to a mixture containing both gyriod () and lamellar () phases. Finite element modelling of the observed phase behaviour during injection indicates that the pressure exerted on the lipid stream during extrusion accounts for the variations in the phase composition of the monoolein:water mixture. Less
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
Intrinsically disordered proteins IDPs can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large reversible protein networks Among these the IDP hub protein G BP forms protein complexes with Caprin and USP and the resulting control of USP activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling However while the identities of protein interactors are known for many of these IDP hub proteins the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood Here we describe in vitro analysis of the ... More
Intrinsically disordered proteins (IDPs) can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large, reversible protein networks. Among these, the IDP hub protein G3BP1 forms protein complexes with Caprin1 and USP10, and the resulting control of USP10 activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling. However, while the identities of protein interactors are known for many of these IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of the G3BP1 affinities, and show that substitution of G3BP1 residues F15 or F33 to tryptophan reduces affinity for both the USP10 and Caprin1 motif peptides. These same mutations significantly reduce formation of complexes by the full-length proteins. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with the USP10 and Caprin1 peptides. An amino-acid scan of the USP10 and Caprin1 motif peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific mutations with the potential to create higher affinity peptides. Taken together, these data show that small changes in 1:1 binding affinity can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. Less
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange altering the on off switch that forms the basis for their signaling functions Using X-ray crystallography nuclear magnetic resonance spectroscopy binding assays and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site ... More
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts. Less
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
L is a potent human monoclonal antibody mAb that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein PfCSP on malaria-infective sporozoites Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development Here we isolate mAbs clonally related to L and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity Pairing the L kappa light chain L with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs cross-react with NANP and more potently neutralize sporozoites in vivo ... More
L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs. Less
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
Structural studies of G-protein-coupled receptors GPCRs are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination During the past decade crystallization in lipidic cubic phase LCP has become the most successful and widely used technique for obtaining such crystals Despite often intense efforts many GPCRs remain refractory to crystallization even if receptors can be purified in sufficient amounts To address this issue we have developed a highly efficient screening and stabilization strategy for GPCRs based on a fluorescence thermal stability assay readout which seems to correlate particularly well with those GPCR constructs that remain native during ... More
Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor. Less
Macrophage migration inhibitory factor MIF is an inflammatory protein with various non-overlapping functions It is not only conserved in mammals but it is found in parasites fish and plants Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro as a catalytic base activates the receptors CD CXCR and CXCR has functional interactions in the cytosol and is reported to be a nuclease There is a solvent channel down its -fold axis with a recently identified gating residue as an allosteric site important for regulating to different extents the enzymatic activity and CD binding and signaling ... More
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight. Less
Pathogen effectors are crucial players during plant colonisation and infection Plant resistance mostly relies on effector recognition to activate defence responses Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment Here we examined the role of genetic diversity of the stem rust Puccinia graminis f sp tritici Pgt AvrSr gene in determining recognition by the corresponding wheat Sr resistance gene We solved the crystal structure of a natural variant of AvrSr and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence We report that AvrSr can ... More
Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment. Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence. We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties. This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations. Less
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
Alphaviruses such as Ross River virus RRV chikungunya virus CHIKV Sindbis virus SINV and Venezuelan equine encephalitis virus VEEV are mosquito-borne pathogens that can cause arthritis or encephalitis diseases Nonstructural protein nsP of alphaviruses possesses RNA-dependent RNA polymerase RdRp activity essential for viral RNA replication No D structure has been available for nsP of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs Here we report crystal structures of the RdRp domain of nsP from both RRV and SINV determined at resolutions of and The structure of the alphavirus RdRp domain appears ... More
Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. Less
Protein homeostasis proteostasis refers to the dynamic regulation of a stable and functional proteome The extensive proteostasis network includes integrated cellular mechanisms that control biogenesis folding trafficking and degradation of proteins Posttranslational modification and protein degradation are key pathways that minimize homeostatic perturbations however disease occurs when these processes become dysregulated Loss of protein homeostasis such as by an increase in misfolded proteins contributes to the pathology of many disorders including cancers to neurodegenerative diseases Maintaining the integrity of the proteome is essential for viability but cells continuously face extracellular and intracellular stresses that destabilize protein homeostasis Collaborations among several ... More
Protein homeostasis (proteostasis) refers to the dynamic regulation of a
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. Less
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. 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
Base excision repair BER is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species BER is initiated by DNA glycosylases each of which repairs a particular class of base damage NTHL a bifunctional DNA glycosylase possesses both glycolytic and -lytic activities with a preference for oxidized pyrimidine substrates Defects in human NTHL drive a class of polyposis colorectal cancer We report the first X-ray crystal structure of hNTHL revealing an open conformation not previously observed in the bacterial orthologs In this conformation the six-helical barrel domain comprising the helix-hairpin-helix HhH DNA binding motif ... More
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1. Less
The COVID- pandemic is having a major impact on public health worldwide and there is an urgent need for the creation of an armamentarium of effective therapeutics including vaccines biologics and small-molecule therapeutics to combat SARS-CoV- and emerging variants Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is an attractive target for therapeutic intervention and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV- -specific antivirals We describe herein the results ... More
The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro. Less
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes Target ubiquitination is achieved through the action of three separate enzymes the E ubiquitin-activating enzyme the E ubiquitin-conjugating enzyme and the E ubiquitin ligase TRIM E ligases are the largest family of RING-type E ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain one or two B-box domains B and B and a coiled-coil domain In addition most TRIMs possess a C-terminal substrate-binding domain which classifies them into one of eleven TRIM classes The ... More
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes. Target ubiquitination is achieved through the action of three separate enzymes; the E1, ubiquitin-activating enzyme, the E2, ubiquitin-conjugating enzyme and the E3, ubiquitin ligase. TRIM E3 ligases are the largest family of RING-type E3 ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain, one or two B-box domains, B1 and B2, and a coiled-coil domain. In addition, most TRIMs possess a C-terminal substrate-binding domain, which classifies them into one of eleven TRIM classes. The PRYSPRY domain is the most common substrate-binding domain in humans and links class IV TRIMs to roles in cellular innate immunity. TRIM22 and TRIM6, are Class IV TRIMs that share high sequence identity with the well-studied HIV restriction factor, TRIM5, and have also been implicated in the anti-viral response. TRIM22 is reported to function directly as a viral restriction factor against RNA viruses such as, IAV, HCV and EMCV. While TRIM6 functions to activate the innate immune signalling pathway through activation of the immune signalling factor, IKK. Aspects of TRIM22 and TRIM6 function remain understudied, including their biochemical and biophysical properties and this is the focus of this study. The results described herein outline key differences in the self-association properties of these proteins in comparison to TRIM5. Furthermore, they highlight discrepancies between the ubiquitination profiles of TRIM22 and TRIM6 presented in the literature and the activity observed in this study. Overall, this emphasizes the need for further study of the roles of TRIM22 and TRIM6, to verify current proposed functions, as well as identify potential additional functions within the cell. Less
Immunomodulatory drugs IMiDs thalidomide lenalidomide and pomalidomide Pom bind to cereblon CRBN and trigger proteasomal degradation of neo-substrates IKZF leading to multiple myeloma MM cell apoptosis Pomalidomide Pom also binds albeit weakly to p -related protein kinase PRPK aka TP RK an understudied kinase reported to be associated with poor prognosis in MM patients Here we developed a series of IMiDs based on Pom and conducted a structure-activity relationship SAR study to identify a potent and selective PRPK binder Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN and suggested specific derivatization to improve affinity ... More
Immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide (Pom) bind to cereblon (CRBN) and trigger proteasomal degradation of neo-substrates IKZF1/3 leading to multiple myeloma (MM) cell apoptosis. Pomalidomide (Pom) also binds albeit weakly to p53-related protein kinase (PRPK, aka TP53RK), an understudied kinase reported to be associated with poor prognosis in MM patients. Here, we developed a series of IMiDs based on Pom and conducted a structure-activity relationship (SAR) study to identify a potent and selective PRPK binder. Structural analysis showed that IMiDs bind PRPK in a fundamentally different way from CRBN, and suggested specific derivatization to improve affinity. We employed a structure-guided strategy to develop compound TXM-02-118, which exhibited nanomolar affinityfor PRPK in binding assays, and showed high selectivity for PRPK over CRBN. Overall, the work represents an initial effort to develop tool compounds for studying PRPK. Moreover, it illustrates how a single class of molecules can use different recognition elements to bind diverse targets using fundamentally different binding poses. This has broad implications for chemical probe and lead compound selectivity profiling, and argues for more wide-spread use of global proteomics or similar methodologies. Less
Immunoglobulin G-based monoclonal antibodies mAbs have become a dominant class of biotherapeutics in recent decades Approved antibodies are mainly of the subclasses IgG IgG and IgG as well as their derivatives Over the decades the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action while the effect on drug product developability has been less thoroughly characterized One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties Several efforts have been made recently to ... More
Immunoglobulin G-based monoclonal antibodies (mAbs) have become a dominant class of biotherapeutics in recent decades. Approved antibodies are mainly of the subclasses IgG1, IgG2, and IgG4, as well as their derivatives. Over the decades, the selection of IgG subclass has frequently been based on the needs of Fc gamma receptor engagement and effector functions for the desired mechanism of action, while the effect on drug product developability has been less thoroughly characterized. One of the major reasons is the lack of systematic understanding of the impact of IgG subclass on the molecular properties. Several efforts have been made recently to compare molecular property differences among these IgG subclasses, but the conclusions from these studies are sometimes obscured by the interference from variable regions. To further establish mechanistic understandings, we conducted a systematic study by grafting three independent variable regions onto human IgG1, an IgG1 variant, IgG2, and an IgG4 variant constant domains and evaluating the impact of subclass and variable regions on their molecular properties. Structural and computational analysis revealed specific molecular features that potentially account for the differential behavior of the IgG subclasses observed experimentally. Our data indicate that IgG subclass plays a significant role on molecular properties, either through direct effects or via the interplay with the variable region, the IgG1 mAbs tend to have higher solubility than either IgG2 or IgG4 mAbs in a common pH 6 buffer matrix, and solution behavior relies heavily on the charge status of the antibody at the desirable pH. Less
Current coronavirus CoV vaccines primarily target immunodominant epitopes in the S subunit which are poorly conserved and susceptible to escape mutations thus threatening vaccine efficacy Here we use structure-guided protein engineering to remove the S subunit from the Middle East respiratory syndrome MERS -CoV spike S glycoprotein and develop stabilized stem SS antigens Vaccination with MERS SS elicits cross-reactive -CoV antibody responses and protects mice against lethal MERS-CoV challenge High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies Among them antibody IgG binds with high affinity to both MERS-CoV ... More
Current coronavirus (CoV) vaccines primarily target immunodominant epitopes in the S1 subunit, which are poorly conserved and susceptible to escape mutations, thus threatening vaccine efficacy. Here, we use structure-guided protein engineering to remove the S1 subunit from the Middle East respiratory syndrome (MERS)-CoV spike (S) glycoprotein and develop stabilized stem (SS) antigens. Vaccination with MERS SS elicits cross-reactive β-CoV antibody responses and protects mice against lethal MERS-CoV challenge. High-throughput screening of antibody-secreting cells from MERS SS-immunized mice led to the discovery of a panel of cross-reactive monoclonal antibodies. Among them, antibody IgG22 binds with high affinity to both MERS-CoV and severe acute respiratory syndrome (SARS)-CoV-2 S proteins, and a combination of electron microscopy and crystal structures localizes the epitope to a conserved coiled-coil region in the S2 subunit. Passive transfer of IgG22 protects mice against both MERS-CoV and SARS-CoV-2 challenge. Collectively, these results provide a proof of principle for cross-reactive CoV antibodies and inform the development of pan-CoV vaccines and therapeutic antibodies. Less
The worldwide outbreak of coronavirus disease COVID- caused by severe acute respiratory syndrome coronavirus SARS-CoV- has become an established global pandemic Alongside vaccines antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID- Here we report the discovery and characterization of PF- an orally bioavailable SARS-CoV- main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles PF- has demonstrated oral activity in a mouse- adapted SARS-CoV- model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase ... More
The worldwide outbreak of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become an established global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to counter the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity, and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse- adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency, in a phase I clinical trial in healthy human participants. Less
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
Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments Here it aids microorganisms to counter osmotic stress and protect their enzymes Ectoine exhibit many interesting properties that is potentially commercially exploitable and it is currently produced and found in several products on the market While ectoine is produced by whole cell synthesis the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally Here we present structural and biochemical characterizations of ectoine synthase from two organisms Chromohalobacter salexigens DSM and Marinobacter sp CK We cloned ... More
Ectoine is a compatible solute found in many microorganisms adapted to survive in saline and other extreme environments. Here, it aids microorganisms to counter osmotic stress and protect their enzymes. Ectoine exhibit many interesting properties that is potentially commercially exploitable, and it is currently produced and found in several products on the market. While ectoine is produced by whole cell synthesis, the EctABC enzymes in the biosynthesis pathway of ectoine was currently not well described structurally or functionally. Here, we present structural and biochemical characterizations of ectoine synthase from two organisms, Chromohalobacter salexigens DSM3043 and Marinobacter sp. CK1. We cloned, expressed and expression optimized both candidates, and purified them by immobilized metal affinity chromatography and gel filtration. C. salexigens EctC (CSEctC) yielded 14-18 mg/L cell culture while Marinobacter sp. CK1 (MarEctC) yielded 0.75-1.5 mg/L culture. We then produced diffracting crystals of CSEctC and obtained a data set from which the structure of CSEctC was determined. We further obtained preliminary biochemical data relating to thermostability and activity from both candidates. The crystal structure from CSEctC shows that it is adapts a typical β-sandwich fold, consistent with earlier structural investigations of other EctC type proteins. This study provides a solid foundation for further research on EctC from our model organisms, and protocols and techniques developed herein can be further optimized to obtain more biochemical data about this interesting enzyme. 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
CD is a tumor necrosis factor TNF receptor which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD Activation of the CD receptor provides a costimulatory signal to promote T cell B cell and NK cell activity to facilitate antitumor and anti-infection immunity Aberrant increased and focused expression of CD on many tumor cells renders CD an attractive therapeutic target for direct tumor killing However despite their use as drug targets to treat cancers the molecular basis and atomic details of CD and CD interaction remain elusive Here we report the crystal structure of human CD in ... More
CD27 is a tumor necrosis factor (TNF) receptor, which stimulates lymphocytes and promotes their differentiation upon activation by TNF ligand CD70. Activation of the CD27 receptor provides a costimulatory signal to promote T cell, B cell, and NK cell activity to facilitate antitumor and anti-infection immunity. Aberrant increased and focused expression of CD70 on many tumor cells renders CD70 an attractive therapeutic target for direct tumor killing. However, despite their use as drug targets to treat cancers, the molecular basis and atomic details of CD27 and CD70 interaction remain elusive. Here we report the crystal structure of human CD27 in complex with human CD70. Analysis of our structure shows that CD70 adopts a classical TNF ligand homotrimeric assembly to engage CD27 receptors in a 3:3 stoichiometry. By combining structural and rational mutagenesis data with reported disease-correlated mutations, we identified the key amino acid residues of CD27 and CD70 that control this interaction. We also report increased potency for plate-bound CD70 constructs compared with solution-phase ligand in a functional activity to stimulate T-cells in vitro. These findings offer new mechanistic insight into this critical costimulatory interaction. Less
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein PrPC a glycosyl-phosphatidylinositol GPI -anchored protein expressed on the cell surface The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein PrPSc with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis Prognoses are devastating with an average survival time of approximately one year after the onset of symptoms Despite the tremendous efforts PrP physiological function and its mechanism of conversion to PrPSc remain elusive This research focuses on Xray crystallographic fragment screening technique to map ... More
Prion diseases result from the ordered accumulation of the misfolded conformer of cellular prion protein (PrPC), a glycosyl-phosphatidylinositol (GPI)-anchored protein expressed on the cell surface. The critical event in prion diseases is the conversion of PrPC into the self-propagating conformer scrapie prion protein, PrPSc, with resultant propagation and accumulation resulting in neuronal death and amyloidogenesis. Prognoses are devastating, with an average survival time of approximately one year after the onset of symptoms. Despite the tremendous efforts, PrP physiological function and its mechanism of conversion to PrPSc remain elusive. This research focuses on Xray crystallographic fragment screening technique to map PrP chemical spaces in order to find lead compounds as part of the drug discovery process. Screening against human PrP, currently stigmatized as an "undruggable" target, can benefit from the fragment screening strategy. This approach relies on low molecular weight compounds to scan the protein surface in search of binding spots in the protein, enhancing the chances of finding ligands that could offer an alternative route to quest a treatment to prion disease. Any hits could be explored to be used for either i) increase PrPC stabilization, increasing the energy barrier for the protein conversion, ii) destabilization, to induce PrP removal from the cell, thus reducing the quantity of PrP available for conversion, or iii) block protein-protein interaction sites between PrPC and PrPSc , inhibiting the conversion process. We have established a reproducible crystal system for which we collected over 1000 X-ray datasets and screened over 600 fragments. Our data shows two ligands interacting with the prion protein and reveal a pyrazole chemical binding motif for an unprecedented small cavity created by a conformational change of the Lys185 sidechain. The in silico analysis of the collected datasets showed that the globular domain of the PrP is unexpectedly rigid. To overcome the difficulty of finding PrP binder molecules, we performed a second fragment screening assay. The second screening was enabled by achieving a more fragment screening-friendly crystal. This search involved screening for a new crystal system, the use of a PrPspecific nanobody, and PEG-based conditions. Our second screening tested over 100 fragments, with no hits. Together, we believe that our work has the potential to provide structural basis to aid the drug discovery regarding the prion protein while also providing an in-depth analysis that can support other X-ray fragment screening endeavors. Less
Medium-chain triglycerides MCTs are an emerging choice to treat neurodegenerative disorders such as Alzheimer s disease They are triesters of glycerol and three medium-chain fatty acids such as capric C and caprylic C acids The availability of C C methyl esters C C ME from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs However there are few reports on enzymes that can efficiently hydrolyse C C ME to industrial specifications Here we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus LTL which hydrolyses C ... More
Medium-chain triglycerides (MCTs) are an emerging choice to treat neurodegenerative disorders such as Alzheimer’s disease. They are triesters of glycerol and three medium-chain fatty acids, such as capric (C8) and caprylic (C10) acids. The availability of C8–C10 methyl esters (C8–C10 ME) from vegetable oil processes has presented an opportunity to use methyl esters as raw materials for the synthesis of MCTs. However, there are few reports on enzymes that can efficiently hydrolyse C8–C10 ME to industrial specifications. Here, we report the discovery and identification of a novel lipase from Lasiodiplodia theobromae fungus (LTL1), which hydrolyses C8–C10 ME efficiently. LTL1 can perform hydrolysis over pH ranges from 3.0 to 9.0 and maintain thermotolerance up to 70 °C. It has high selectivity for monoesters over triesters and displays higher activity over commercially available lipases for C8–C10 ME to achieve 96.17% hydrolysis within 31 h. Structural analysis by protein X-ray crystallography revealed LTL1’s well-conserved lipase core domain, together with a partially resolved N-terminal subdomain and an inserted loop, which may suggest its hydrolytic preference for monoesters. In conclusion, our results suggest that LTL1 provides a tractable route towards to production of C8–C10 fatty acids from methyl esters for the synthesis of MCTs. Less
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV- envelope glycoprotein Env is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies NAbs Here we analyzed monoclonal antibodies mAbs isolated from non-human primates NHPs immunized with variants of a native flexibly linked NFL HIV- Env stabilized trimer derived from the tier clade C strain The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from to g ml IC Structural characterization using negative-stain EM and X-ray crystallography identified the variable region V of the NFL trimer to be the common ... More
Understanding the molecular mechanisms by which antibodies target and neutralize the HIV-1 envelope glycoprotein (Env) is critical in guiding immunogen design and vaccine development aimed at eliciting cross-reactive neutralizing antibodies (NAbs). Here, we analyzed monoclonal antibodies (mAbs) isolated from non-human primates (NHPs) immunized with variants of a native flexibly linked (NFL) HIV-1 Env stabilized trimer derived from the tier 2 clade C 16055 strain. The antibodies displayed neutralizing activity against the autologous virus with potencies ranging from 0.005 to 3.68 μg/ml (IC50). Structural characterization using negative-stain EM and X-ray crystallography identified the variable region 2 (V2) of the 16055 NFL trimer to be the common epitope for these antibodies. The crystal structures revealed that the V2 segment adopts a β-hairpin motif identical to that observed in the 16055 NFL crystal structure. These results depict how vaccine-induced antibodies derived from different clonal lineages penetrate through the glycan shield to recognize a hypervariable region within V2 (residues 184–186) that is unique to the 16055 strain. They also provide potential explanations for the potent autologous neutralization of these antibodies, confirming the immunodominance of this site and revealing that multiple angles of approach are permissible for affinity/avidity that results in potent neutralizing capacity. The structural analysis reveals that the most negatively charged paratope correlated with the potency of the mAbs. The atomic level information is of interest to both define the means of autologous neutralization elicited by different tier 2-based immunogens and facilitate trimer redesign to better target more conserved regions of V2 to potentially elicit cross-neutralizing HIV-1 antibodies. Less
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type secretion system T SS and target leukocytes to subvert host defenses T SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T SS and drives secretion via a Brownian ratchet mechanism Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin an RTX leukotoxin essential for B pertussis colonization have been shown to target the RTX domain and prevent binding to the M integrin receptor Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform ... More
RTX leukotoxins are a diverse family of prokaryotic virulence factors that are secreted by the type 1 secretion system (T1SS) and target leukocytes to subvert host defenses. T1SS substrates all contain a C-terminal RTX domain that mediates recruitment to the T1SS and drives secretion via a Brownian ratchet mechanism. Neutralizing antibodies against the Bordetella pertussis adenylate cyclase toxin, an RTX leukotoxin essential for B. pertussis colonization, have been shown to target the RTX domain and prevent binding to the αMβ2 integrin receptor. Knowledge of the mechanisms by which antibodies bind and neutralize RTX leukotoxins is required to inform structure-based design of bacterial vaccines, however, no structural data are available for antibody binding to any T1SS substrate. Here, we determine the crystal structure of an engineered RTX domain fragment containing the αMβ2-binding site bound to two neutralizing antibodies. Notably, the receptor-blocking antibodies bind to the linker regions of RTX blocks I–III, suggesting they are key neutralization-sensitive sites within the RTX domain and are likely involved in binding the αMβ2 receptor. As the engineered RTX fragment contained these key epitopes, we assessed its immunogenicity in mice and showed that it elicits similar neutralizing antibody titers to the full RTX domain. The results from these studies will support the development of bacterial vaccines targeting RTX leukotoxins, as well as next-generation B. pertussis vaccines. Less
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
The macrophage migration inhibitory factor MIF family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism s Previously we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel Despite structural and functional similarity the MIF homolog D-dopachrome tautomerase also called MIF- has low sequence identity prompting the question of whether this dynamic regulatory network is conserved Here we establish the structural basis of an allosteric site in MIF- showing with solution NMR that dynamic communication is preserved in MIF- despite differences in the primary sequence X-ray crystallography ... More
The macrophage migration inhibitory factor (MIF) family of cytokines contains multiple ligand-binding sites and mediates immunomodulatory processes through an undefined mechanism(s). Previously, we reported a dynamic relay connecting the MIF catalytic site to an allosteric site at its solvent channel. Despite structural and functional similarity, the MIF homolog D-dopachrome tautomerase (also called MIF-2) has low sequence identity (35%), prompting the question of whether this dynamic regulatory network is conserved. Here, we establish the structural basis of an allosteric site in MIF-2, showing with solution NMR that dynamic communication is preserved in MIF-2 despite differences in the primary sequence. X-ray crystallography and NMR detail the structural consequences of perturbing residues in this pathway, which include conformational changes surrounding the allosteric site, despite global preservation of the MIF-2 fold. Molecular simulations reveal MIF-2 to contain a comparable hydrogen bond network to that of MIF, which was previously hypothesized to influence catalytic activity by modulating the strength of allosteric coupling. Disruption of the allosteric relay by mutagenesis also attenuates MIF-2 enzymatic activity in vitro and the activation of the cluster of differentiation 74 receptor in vivo, highlighting a conserved point of control for nonoverlapping functions in the MIF superfamily. Less