344 Citations
The zinc-finger transcription factor Helios is critical for maintaining the identity anergic phenotype and suppressive activity of regulatory T Treg cells While it is an attractive target to enhance the efficacy of currently approved immunotherapies no existing approaches can directly modulate Helios activity or abundance Here we report the structure-guided development of small molecules that recruit the E ubiquitin ligase substrate receptor cereblon to Helios thereby promoting its degradation Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells establishing a route towards Helios-targeting therapeutics More generally this study provides a framework for the development ... More
The zinc-finger transcription factor Helios is critical for maintaining the identity, anergic phenotype and suppressive activity of regulatory T (Treg) cells. While it is an attractive target to enhance the efficacy of currently approved immunotherapies, no existing approaches can directly modulate Helios activity or abundance. Here, we report the structure-guided development of small molecules that recruit the E3 ubiquitin ligase substrate receptor cereblon to Helios, thereby promoting its degradation. Pharmacological Helios degradation destabilized the anergic phenotype and reduced the suppressive activity of Treg cells, establishing a route towards Helios-targeting therapeutics. More generally, this study provides a framework for the development of small-molecule degraders for previously unligandable targets by reprogramming E3 ligase substrate specificity. Less
The leukotriene B receptor BLT regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious allergic metabolic and tumorigenic human diseases Here we present a crystal structure of human BLT hBLT in complex with a selective antagonist MK-D- developed for the treatment of type diabetes and other inflammatory conditions Comprehensive analysis of the structure and structure-activity relationship data reinforced by site-directed mutagenesis and docking studies reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species The structure helps to identify a putative membrane-buried ligand access channel ... More
The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design. Less
The adhesion G protein coupled receptor CD and its ligand complement decay-accelerating factor CD are important binding partners in the human immune system Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis as well as various cancers Previous literatures have indicated that the CD includes to epidermal growth factor EGF domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat SCR domains of CD However the details of this interaction remain elusive especially why the CD binds with the highest affinity to the shortest isoform ... More
The adhesion G protein–coupled receptor CD97 and its ligand complement decay-accelerating factor CD55 are important binding partners in the human immune system. Dysfunction in this binding has been linked to immune disorders such as multiple sclerosis and rheumatoid arthritis, as well as various cancers. Previous literatures have indicated that the CD97 includes 3 to 5 epidermal growth factor (EGF) domains at its N terminus and these EGF domains can bind to the N-terminal short consensus repeat (SCR) domains of CD55. However, the details of this interaction remain elusive, especially why the CD55 binds with the highest affinity to the shortest isoform of CD97 (EGF1,2,5). Herein, we designed a chimeric expression construct with the EGF1,2,5 domains of CD97 and the SCR1–4 domains of CD55 connected by a flexible linker and determined the complex structure by crystallography. Our data reveal that the two proteins adopt an overall antiparallel binding mode involving the SCR1–3 domains of CD55 and all three EGF domains of CD97. Mutagenesis data confirmed the importance of EGF5 in the interaction and explained the binding specificity between CD55 and CD97. The architecture of CD55–CD97 binding mode together with kinetics suggests a force-resisting shearing stretch geometry when forces applied to the C termini of both proteins in the circulating environment. The potential of the CD55–CD97 complex to withstand tensile force may provide a basis for the mechanosensing mechanism for activation of adhesion G protein–coupled receptors. Less
Human parainfluenza virus type III HPIV is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations including the immunocompromised There are currently no effective vaccines or therapeutics available resulting in tens of thousands of hospitalizations per year In an effort to discover a protective antibody against HPIV we screened the B cell repertoires from peripheral blood tonsils and spleen from healthy children and adults These analyses yielded five monoclonal antibodies that potently neutralized HPIV in vitro These HPIV -neutralizing antibodies targeted two non-overlapping epitopes of the HPIV F protein with most targeting the apex Prophylactic ... More
Human parainfluenza virus type III (HPIV3) is a common respiratory pathogen that afflicts children and can be fatal in vulnerable populations, including the immunocompromised. There are currently no effective vaccines or therapeutics available, resulting in tens of thousands of hospitalizations per year. In an effort to discover a protective antibody against HPIV3, we screened the B cell repertoires from peripheral blood, tonsils, and spleen from healthy children and adults. These analyses yielded five monoclonal antibodies that potently neutralized HPIV3 in vitro. These HPIV3-neutralizing antibodies targeted two non-overlapping epitopes of the HPIV3 F protein, with most targeting the apex. Prophylactic administration of one of these antibodies, PI3-E12, resulted in potent protection against HPIV3 infection in cotton rats. Additionally, PI3-E12 could also be used therapeutically to suppress HPIV3 in immunocompromised animals. These results demonstrate the potential clinical utility of PI3-E12 for the prevention or treatment of HPIV3 in both immunocompetent and immunocompromised individuals. Less
Alginate is the structural polysaccharide of the cell wall of brown algae which is an important carbon source for marine life The depolymerization of alginate is dependent on alginate lyases Recent studies showed that the alginate utilization ability had been obtained by human gut microbes In contrast to the great number of studies on alginate lyases from marine soil organisms studies on alginate lyases from gut microbes are still limited Here the structure of a polysaccharide lyase family PL alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography which represents the cluster of two-domain PL alginate ... More
Alginate is the structural polysaccharide of the cell wall of brown algae, which is an important carbon source for marine life. The depolymerization of alginate is dependent on alginate lyases. Recent studies showed that the alginate utilization ability had been obtained by human gut microbes. In contrast to the great number of studies on alginate lyases from marine/soil organisms, studies on alginate lyases from gut microbes are still limited. Here, the structure of a polysaccharide lyase family 6 (PL6) alginate lyase from human gut microbe Bacteroides clarus was solved by X-ray crystallography, which represents the cluster of two-domain PL6 alginate lyases from Bacteroidetes. Similar with the two-domain alginate lyase AlyGC originated from marine bacterium, both the N terminal domain (NTD) and C terminal domain (CTD) of BcAlyPL6 show right-handed parallel β-helix fold. However, unlike AlyGC, which forms a homodimer, BcAlyPL6 functions as a monomer. Biochemical analysis indicates that the substrate binding affinity is mainly contributed by the NTD while the CTD of BcAlyPL6 is involved in the formation of −1 subsite, which is essential for substrate turnover rate. Furthermore, CTD is involved in shaping a closed catalytic pocket, and deletion of it leads to increased activity towards highly polymerized substrate. Structure comparison of PL6 family alginate lyases implies that the linkers of two-domain alginate lyases might have evolutionary relationship with the N/C terminal extension of single-domain lyases. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
Light-oxygen-voltage LOV domains are widespread photosensory modules that can be used in fluorescence microscopy optogenetics and controlled production of reactive oxygen species All of the currently known LOV domains have absorption maxima in the range of to nm and it is not clear whether they can be shifted significantly using mutations Here we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP Gln to asparagine aspartate glutamate histidine lysine and arginine Absorption spectra of all of the mutants are blue-shifted with the maximal shift of nm ... More
Light-oxygen-voltage (LOV) domains are widespread photosensory modules that can be used in fluorescence microscopy, optogenetics and controlled production of reactive oxygen species. All of the currently known LOV domains have absorption maxima in the range of ~440 to ~450 nm, and it is not clear whether they can be shifted significantly using mutations. Here, we have generated a panel of LOV domain variants by mutating the key chromophore-proximal glutamine aminoacid of a thermostable flavin based fluorescent protein CagFbFP (Gln148) to asparagine, aspartate, glutamate, histidine, lysine and arginine. Absorption spectra of all of the mutants are blue-shifted, with the maximal shift of 8 nm observed for the Q148H variant. While CagFbFP and its Q148N/D/E variants are not sensitive to pH, Q148H/K/R reveal a moderate red shift induced byacidic pH. To gain further insight, we determined high resolution crystal structures of all of the mutants studied at the resolutions from 1.07 Å for Q148D to 1.63 Å for Q148R. Whereas in some of the variants, the aminoacid 148 remains in the vicinity of the flavin, in Q148K, Q148R and partially Q148D, the C-terminus of the protein unlatches and the side chain of the residue 148 is reoriented away from the chromophore. Our results explain the absence of color shifts from replacing Gln148 with charged aminoacids and pave the way for rational design of color-shifted flavin based fluorescent proteins. Less
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution yet the unique contributions of multimeric IgM antibodies to infection control are not well understood This is partially due to the difficulty of distinguishing low-affinity IgM secreted rapidly by plasmablasts from high-affinity antibodies derived from later-arising memory cells We developed a pipeline to express B cell receptors BCRs from Plasmodium falciparum specific IgM and IgG human memory B cells MBCs as both IgM and IgG molecules BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity Crystallization of one IgM MBC-derived antibody complexed with antigen defined a linear epitope within ... More
Multimeric immunoglobulin-like molecules arose early in vertebrate evolution, yet the unique contributions of multimeric IgM antibodies to infection control are not well understood. This is partially due to the difficulty of distinguishing low-affinity IgM, secreted rapidly by plasmablasts, from high-affinity antibodies derived from later-arising memory cells. We developed a pipeline to express B cell receptors (BCRs) from Plasmodium falciparum–specific IgM+ and IgG+ human memory B cells (MBCs) as both IgM and IgG molecules. BCRs from both subsets were somatically hypermutated and exhibited comparable monomeric affinity. Crystallization of one IgM+ MBC-derived antibody complexed with antigen defined a linear epitope within a conserved Plasmodium protein. In its physiological multimeric state, this antibody displayed exponentially higher antigen binding than a clonally identical IgG monomer, and more effectively inhibited P. falciparum invasion. Forced multimerization of this IgG significantly improved both antigen binding and parasite restriction, underscoring how avidity can alter antibody function. This work demonstrates the potential of high-avidity IgM in both therapeutics and vaccines. Less
Mutations in two different domains of the ubiquitously expressed TRIM protein give rise to two clinically separate diseases one of which is Limb-girdle muscular dystrophy type H LGMD H Uncovering the muscle-specific role of TRIM in LGMD H pathogenesis has proven difficult as neurogenic phenotypes independent of LGMD H pathology are present in TRIM KO mice We previously established a platform to study LGMD H pathogenesis using Drosophila melanogaster as a model Here we show that LGMD H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM Furthermore transgenic expression of a subset ... More
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration. Less
The Parkin co-regulated gene protein PACRG binds at the inner junction between doublet microtubules of the axoneme a structure found in flagella and cilia PACRG binds to the adaptor protein meiosis expressed gene MEIG but how they bind to microtubules is unknown Here we report the crystal structure of human PACRG in complex with MEIG PACRG adopts a helical repeat fold with a loop that interacts with MEIG Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation ... More
The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA Here we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides Kinetic NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension and in part because of the greater distance between the attacking RNA primer -hydroxyl and the phosphate of the incoming threo-nucleotide ... More
The prebiotic synthesis of ribonucleotides is likely to have been accompanied by the synthesis of noncanonical nucleotides including the threo-nucleotide building blocks of TNA. Here, we examine the ability of activated threo-nucleotides to participate in nonenzymatic template-directed polymerization. We find that primer extension by multiple sequential threo-nucleotide monomers is strongly disfavored relative to ribo-nucleotides. Kinetic, NMR and crystallographic studies suggest that this is due in part to the slow formation of the imidazolium-bridged TNA dinucleotide intermediate in primer extension, and in part because of the greater distance between the attacking RNA primer 3′-hydroxyl and the phosphate of the incoming threo-nucleotide intermediate. Even a single activated threo-nucleotide in the presence of an activated downstream RNA oligonucleotide is added to the primer 10-fold more slowly than an activated ribonucleotide. In contrast, a single activated threo-nucleotide at the end of an RNA primer or in an RNA template results in only a modest decrease in the rate of primer extension, consistent with the minor and local structural distortions revealed by crystal structures. Our results are consistent with a model in which heterogeneous primordial oligonucleotides would, through cycles of replication, have given rise to increasingly homogeneous RNA strands. Less
White spot syndrome virus WSSV the causative agent of white spot disease WSD severely affecting crustacean life forms is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target leading to various anti-cancer drugs The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase wTS Here we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate dUMP and methotrexate at a resolution of and ... More
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- and other SARS-like-CoVs encode tandem macrodomains within non-structural protein nsp The first macrodomain Mac is conserved throughout CoVs and binds to and hydrolyzes mono-ADP-ribose MAR from target proteins Mac likely counters host-mediated anti-viral ADP-ribosylation a posttranslational modification that is part of the host response to viral infections Mac is essential for pathogenesis in multiple animal models of CoV infection implicating it as a virulence factor and potential therapeutic target Here we report the crystal structure of SARS-CoV- Mac in complex with ADP-ribose SARS-CoV- SARS-CoV and MERS-CoV Mac exhibit similar structural folds and all proteins ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity. Less
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork In eukaryotes proliferating cell nuclear antigen PCNA plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability PCNA from several organisms including Saccharomyces cerevisiae has been structurally characterised However the structural analyses of PCNA from fungal pathogens are limited Recently we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S cerevisiae Still it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic ... More
An assembly of multiprotein complexes achieves chromosomal DNA replication at the replication fork. In eukaryotes, proliferating cell nuclear antigen (PCNA) plays a vital role in the assembly of multiprotein complexes at the replication fork and is essential for cell viability. PCNA from several organisms, including Saccharomyces cerevisiae, has been structurally characterised. However, the structural analyses of PCNA from fungal pathogens are limited. Recently, we have reported that PCNA from the opportunistic fungal pathogen Candida albicans complements the essential functions of ScPCNA in S. cerevisiae. Still, it only partially rescues the loss of ScPCNA when the yeast cells are under genotoxic stress. To understand this further, herein, we have determined the crystal structure of CaPCNA and compared that with the existing structures of other fungal and human PCNA. Our comparative structural and in-solution small-angle X-ray scattering (SAXS) analyses reveal that CaPCNA forms a stable homotrimer, both in crystal and in solution. It displays noticeable structural alterations in the oligomerisation interface, P-loop and hydrophobic pocket regions, suggesting its differential function in a heterologous system and avenues for developing specific therapeutics. Less
Catalysis of human phosphoglycerate mutase is dependent on a -bisphosphoglycerate cofactor dPGM whereas the nonhomologous isozyme in many parasitic species is cofactor independent iPGM This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms We previously discovered ipglycermide a potent inhibitor of iPGM from a large combinatorial cyclic peptide library To fully delineate the ipglycermide pharmacophore herein we construct a detailed structure activity relationship using substituted ipglycermide analogs Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM measured as fold enrichment relative to the index residue by deep sequencing of an mRNA ... More
Catalysis of human phosphoglycerate mutase is dependent on a 2,3-bisphosphoglycerate cofactor (dPGM), whereas the nonhomologous isozyme in many parasitic species is cofactor independent (iPGM). This mechanistic and phylogenetic diversity offers an opportunity for selective pharmacologic targeting of glycolysis in disease-causing organisms. We previously discovered ipglycermide, a potent inhibitor of iPGM, from a large combinatorial cyclic peptide library. To fully delineate the ipglycermide pharmacophore, herein we construct a detailed structure–activity relationship using 280 substituted ipglycermide analogs. Binding affinities of these analogs to immobilized Caenorhabditis elegans iPGM, measured as fold enrichment relative to the index residue by deep sequencing of an mRNA display library, illuminated the significance of each amino acid to the pharmacophore. Using cocrystal structures and binding kinetics, we show that the high affinity of ipglycermide for iPGM orthologs, from Brugia malayi, Onchocerca volvulus, Dirofilaria immitis, and Escherichia coli, is achieved by a codependence between (1) the off-rate mediated by the macrocycle Cys14 thiolate coordination to an active-site Zn2+ in the iPGM phosphatase domain and (2) shape complementarity surrounding the macrocyclic core at the phosphotransferase–phosphatase domain interface. Our results show that the high-affinity binding of ipglycermide to iPGMs freezes these structurally dynamic enzymes into an inactive, stable complex. Less
Electron crystallography of sub-micrometre-sized D protein crystals has emerged recently as a valuable field of structural biology In meso crystallization methods utilizing lipidic mesophases particularly lipidic cubic phases LCPs can produce high-quality D crystals of membrane proteins MPs A major step towards realizing D electron crystallography of MP crystals grown in meso is to demonstrate electron diffraction from such crystals The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals Additionally the crystals have to be thin enough to let electrons traverse them without significant multiple scattering In the present ... More
Electron crystallography of sub-micrometre-sized 3D protein crystals has emerged recently as a valuable field of structural biology. In meso crystallization methods, utilizing lipidic mesophases, particularly lipidic cubic phases (LCPs), can produce high-quality 3D crystals of membrane proteins (MPs). A major step towards realizing 3D electron crystallography of MP crystals, grown in meso, is to demonstrate electron diffraction from such crystals. The first task is to remove the viscous and sticky lipidic matrix that surrounds the crystals without damaging the crystals. Additionally, the crystals have to be thin enough to let electrons traverse them without significant multiple scattering. In the present work, the concept that focused ion beam milling at cryogenic temperatures (cryo-FIB milling) can be used to remove excess host lipidic mesophase matrix is experimentally verified, and then the crystals are thinned to a thickness suitable for electron diffraction. In this study, bacteriorhodopsin (BR) crystals grown in a lipidic cubic mesophase of monoolein were used as a model system. LCP from a part of a hexagon-shaped plate-like BR crystal (∼10 µm in thickness and ∼70 µm in the longest dimension), which was flash-frozen in liquid nitrogen, was milled away with a gallium FIB under cryogenic conditions, and a part of the crystal itself was thinned into a ∼210 nm-thick lamella with the ion beam. The frozen sample was then transferred into an electron cryo-microscope, and a nanovolume of ∼1400 × 1400 × 210 nm of the BR lamella was exposed to 200 kV electrons at a fluence of ∼0.06 e Å−2. The resulting electron diffraction peaks were detected beyond 2.7 Å resolution (with an average peak height to background ratio of >2) by a CMOS-based Ceta 16M camera. The results demonstrate that cryo-FIB milling produces high-quality lamellae from crystals grown in lipidic mesophases and pave the way for 3D electron crystallography on crystals grown or embedded in highly viscous media. Less
Structural studies of challenging targets such as G protein-coupled receptors GPCRs have accelerated during the last several years due to the development of new approaches including small-wedge and serial crystallography Here we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase LCP grown microcrystals of two human GPCRs Cysteinyl leukotriene receptors and CysLT R and CysLT R in complex with various antagonists Five datasets were collected using small-wedge synchrotron crystallography SWSX at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions Two datasets were collected using X-ray free electron laser XFEL serial ... More
Structural studies of challenging targets such as G protein-coupled receptors (GPCRs) have accelerated during the last several years due to the development of new approaches, including small-wedge and serial crystallography. Here, we describe the deposition of seven datasets consisting of X-ray diffraction images acquired from lipidic cubic phase (LCP) grown microcrystals of two human GPCRs, Cysteinyl leukotriene receptors 1 and 2 (CysLT1R and CysLT2R), in complex with various antagonists. Five datasets were collected using small-wedge synchrotron crystallography (SWSX) at the European Synchrotron Radiation Facility with multiple crystals under cryo-conditions. Two datasets were collected using X-ray free electron laser (XFEL) serial femtosecond crystallography (SFX) at the Linac Coherent Light Source, with microcrystals delivered at room temperature into the beam within LCP matrix by a viscous media microextrusion injector. All seven datasets have been deposited in the open-access databases Zenodo and CXIDB. Here, we describe sample preparation and annotate crystallization conditions for each partial and full datasets. We also document full processing pipelines and provide wrapper scripts for SWSX and SFX data processing. Less
Excitatory amino acid transporters EAATs harness Na K and H gradients for fast and efficient glutamate removal from the synaptic cleft Since each glutamate is cotransported with three Na ions Na gradients are the predominant driving force for glutamate uptake We combined all-atom molecular dynamics simulations fluorescence spectroscopy and x-ray crystallography to study Na substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type Na -only bound GltPh at - resolution revealed the fully open outward-facing state primed for subsequent substrate binding Simulations and kinetic experiments established that only the binding of two Na ... More
Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh. A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations. Less
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly Vertebrate transferrins coordinate iron through interactions with two tyrosines an aspartate a histidine and a carbonate anion and conformational changes that occur upon iron binding and release have been described Much less is known about the structure and functions of insect transferrin- Tsf which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf differs from that of the vertebrate transferrins Here we report the first crystal structure resolution of an ... More
Transferrins function in iron sequestration and iron transport by binding iron tightly and reversibly. Vertebrate transferrins coordinate iron through interactions with two tyrosines, an aspartate, a histidine, and a carbonate anion, and conformational changes that occur upon iron binding and release have been described. Much less is known about the structure and functions of insect transferrin-1 (Tsf1), which is present in hemolymph and influences iron homeostasis mostly by unknown mechanisms. Amino acid sequence and biochemical analyses have suggested that iron coordination by Tsf1 differs from that of the vertebrate transferrins. Here we report the first crystal structure (2.05 Å resolution) of an insect transferrin. Manduca sexta (MsTsf1) in the holo form exhibits a bilobal fold similar to that of vertebrate transferrins, but its carboxyl-lobe adopts a novel orientation and contacts with the amino-lobe. The structure revealed coordination of a single Fe3+ ion in the amino-lobe through Tyr90, Tyr204, and two carbonate anions. One carbonate anion is buried near the ferric ion and is coordinated by four residues, whereas the other carbonate anion is solvent exposed and coordinated by Asn121. Notably, these residues are highly conserved in Tsf1 orthologs. Docking analysis suggested that the solvent exposed carbonate position is capable of binding alternative anions. These findings provide a structural basis for understanding Tsf1 function in iron sequestration and transport in insects as well as insight into the similarities and differences in iron homeostasis between insects and humans. Less
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families Here we present a functional and structural characterization of two proteins of viral rhodopsin group OLPVR and VirChR Functional analysis of VirChR shows that it is a highly selective Na K -conducting channel and in contrast to known cation channelrhodopsins it is impermeable to Ca ions We show that upon illumination VirChR is ... More
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na+/K+-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca2+ ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca2+ permeability. Less
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan As Lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a ... More
Bacteria are surrounded by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide-peptide precursor called Lipid II is polymerized and crosslinked to produce mature peptidoglycan. As Lipid II is polymerized, nascent polymers remain membrane-anchored at one end and the other end becomes crosslinked to the matrix2–4. A longstanding question is how bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan. Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein containing eight transmembrane helices form a complex that acts as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A 2.6 Å resolution structure of the complex shows that the membrane protein scaffolds the hydrolase to orient its active site for cleavage of the glycan strand. We propose that this complex serves to detach newly-synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival During cell wall assembly a lipid-linked disaccharide peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan As lipid II is polymerized nascent polymers remain membrane-anchored at one end and the other end becomes cross-linked to the matrix How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question Here we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may ... More
Bacteria are encapsulated by a peptidoglycan cell wall that is essential for their survival1. During cell wall assembly, a lipid-linked disaccharide–peptide precursor called lipid II is polymerized and cross-linked to produce mature peptidoglycan. As lipid II is polymerized, nascent polymers remain membrane-anchored at one end, and the other end becomes cross-linked to the matrix2,3,4. How bacteria release newly synthesized peptidoglycan strands from the membrane to complete the synthesis of mature peptidoglycan is a long-standing question. Here, we show that a Staphylococcus aureus cell wall hydrolase and a membrane protein that contains eight transmembrane helices form a complex that may function as a peptidoglycan release factor. The complex cleaves nascent peptidoglycan internally to produce free oligomers as well as lipid-linked oligomers that can undergo further elongation. The polytopic membrane protein, which is similar to a eukaryotic CAAX protease, controls the length of these products. A structure of the complex at a resolution of 2.6 Å shows that the membrane protein scaffolds the hydrolase to orient its active site for cleaving the glycan strand. We propose that this complex functions to detach newly synthesized peptidoglycan polymer from the cell membrane to complete integration into the cell wall matrix. Less
- - protein isoforms regulate multiple processes in eukaryotes including apoptosis and cell division - - proteins preferentially recognize phosphorylated unstructured motifs justifying the protein-peptide binding approach to study - - phosphotarget complexes Tethering of human - - with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides simultaneously facilitating purification and crystallization Nevertheless the broader applicability to other - - isoforms and phosphopeptides was unclear Here we designed a novel - - chimera with a conserved phosphopeptide from BAD whose complex with - - is a gatekeeper of apoptosis regulation The ... More
14-3-3 protein isoforms regulate multiple processes in eukaryotes, including apoptosis and cell division. 14-3-3 proteins preferentially recognize phosphorylated unstructured motifs, justifying the protein-peptide binding approach to study 14-3-3/phosphotarget complexes. Tethering of human 14-3-3σ with partner phosphopeptides via a short linker has provided structural information equivalent to the use of synthetic phosphopeptides, simultaneously facilitating purification and crystallization. Nevertheless, the broader applicability to other 14-3-3 isoforms and phosphopeptides was unclear. Here, we designed a novel 14-3-3ζ chimera with a conserved phosphopeptide from BAD, whose complex with 14-3-3 is a gatekeeper of apoptosis regulation. The chimera could be bacterially expressed and purified without affinity tags. Co-expressed PKA efficiently phosphorylates BAD within the chimera and blocks its interaction with a known 14-3-3 phosphotarget, suggesting occupation of the 14-3-3 grooves by the tethered BAD phosphopeptide. Efficient crystallization of the engineered protein suggests suitability of the “chimeric” approach for studies of other relevant 14-3-3 complexes. Less
SARS-CoV- is a betacoronavirus virus responsible for the COVID- pandemic Here we determined the X-ray crystal structure of a potent neutralizing monoclonal antibody CV isolated from a patient infected with SARS-CoV- in complex with the receptor binding domain RBD The structure reveals CV s epitope overlaps with the human ACE receptor binding site thus providing the structural basis for its neutralization by preventing ACE binding
Cation-chloride cotransporters CCCs regulate the movement of chloride across membranes controlling physiological processes from cell volume maintenance to neuronal signaling Human CCCs are clinical targets for existing diuretics and potentially additional indications Here we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter Caenorhabditis elegans KCC- We observe a core a fold conserved among CCCs Using structure-based sequence alignment we analyze similarities and differences to the C-terminal domains of other CCC family members We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved suggesting ... More
Cation-chloride cotransporters (CCCs) regulate the movement of chloride across membranes, controlling physiological processes from cell volume maintenance to neuronal signaling. Human CCCs are clinical targets for existing diuretics and potentially additional indications. Here, we report the X-ray crystal structure of the soluble C-terminal regulatory domain of a eukaryotic potassium-chloride cotransporter, Caenorhabditis elegans KCC-1. We observe a core a/� fold conserved among CCCs. Using structure-based sequence alignment, we analyze similarities and differences to the C-terminal domains of other CCC family members. We find that important regulatory motifs are in less-structured regions and residues important for dimerization are not widely conserved, suggesting that oligomerization and its effects may vary within the larger family. This snapshot of a eukaryotic KCC is a valuable starting point for the rational design of studies of cellular chloride regulation. Less
Pathogenic coronaviruses are a major threat to global public health as exemplified by severe acute respiratory syndrome coronavirus SARS-CoV Middle East respiratory syndrome coronavirus MERS-CoV and the newly emerged SARS-CoV- the causative agent of coronavirus disease COVID- We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus C-like protease CLpro an enzyme essential for viral replication The optimized compounds were effective against several human coronaviruses including MERS-CoV SARS-CoV and SARS-CoV- in an enzyme assay and in cell-based assays using Huh- and Vero E cell lines Two selected compounds showed antiviral effects against SARS-CoV- in cultured primary ... More
Pathogenic coronaviruses are a major threat to global public health, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and the newly emerged SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). We describe herein the structure-guided optimization of a series of inhibitors of the coronavirus 3C-like protease (3CLpro), an enzyme essential for viral replication. The optimized compounds were effective against several human coronaviruses including MERS-CoV, SARS-CoV, and SARS-CoV-2 in an enzyme assay and in cell-based assays using Huh-7 and Vero E6 cell lines. Two selected compounds showed antiviral effects against SARS-CoV-2 in cultured primary human airway epithelial cells. In a mouse model of MERS-CoV infection, administration of a lead compound 1 day after virus infection increased survival from 0 to 100% and reduced lung viral titers and lung histopathology. These results suggest that this series of compounds has the potential to be developed further as antiviral drugs against human coronaviruses. Less
Ghrelin is a gastric peptide hormone with important physiological functions The unique feature of ghrelin is its Serine acyl-modification which is essential for ghrelin s activity However it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity To address these questions we solved the crystal structure of the ghrelin receptor bound to antagonist The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E and R A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap crevasse between the TM and TM bundles that is rich in ... More
Ghrelin is a gastric peptide hormone with important physiological functions. The unique feature of ghrelin is its Serine 3 acyl-modification, which is essential for ghrelin’s activity. However, it remains to be elucidated why the acyl-modification of ghrelin is necessary for activity. To address these questions, we solved the crystal structure of the ghrelin receptor bound to antagonist. The ligand-binding pocket of the ghrelin receptor is bifurcated by a salt bridge between E124 and R283. A striking feature of the ligand-binding pocket of the ghrelin receptor is a wide gap (crevasse) between the TM6 and TM7 bundles that is rich in hydrophobic amino acids, including a cluster of phenylalanine residues. Mutagenesis analyses suggest that the interaction between the gap structure and the acyl acid moiety of ghrelin may participate in transforming the ghrelin receptor into an active conformation. Less
G-protein-coupled receptors GPCRs the largest family of cell-surface membrane proteins mediate the intracellular signal transduction of many external ligands Thus GPCRs have become important drug targets X-ray crystal structures of GPCRs are very useful for structure-based drug design SBDD Herein we produced a new antibody SRP targeting the thermostabilised apocytochrome b from Escherichia coli M W H I R L BRIL We found that a fragment of this antibody SRP Fab facilitated the crystallisation of the BRIL-tagged ligand bound GPCRs HT B and AT R Furthermore the electron densities of the ligands were resolved suggesting that SPR Fab is versatile ... More
G-protein-coupled receptors (GPCRs)�the largest family of cell-surface membrane proteins�mediate the intracellular signal transduction of many external ligands. Thus, GPCRs have become important drug targets. X-ray crystal structures of GPCRs are very useful for structure-based drug design (SBDD). Herein, we produced a new antibody (SRP2070) targeting the thermostabilised apocytochrome b562 from Escherichia coli M7W/H102I/R106L (BRIL). We found that a fragment of this antibody (SRP2070Fab) facilitated the crystallisation of the BRIL-tagged, ligand bound GPCRs, 5HT1B and AT2R. Furthermore, the electron densities of the ligands were resolved, suggesting that SPR2070Fab is versatile and adaptable for GPCR SBDD. We anticipate that this new tool will significantly accelerate structure determination of other GPCRs and the design of small molecular drugs targeting them. Less
Emergent nanoscale order in materials such as self-assembled lipid phases colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering SAXS Frequently residual disorder in these materials prevents high-resolution D structural characterization Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local D structure via a rich real-space distribution of three- and four-body statistics We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid revealing non-uniform curvature in the lipid channels likely due to phase separation of the lipids in the membrane Our many-body technique has general ... More
Emergent nanoscale order in materials such as self-assembled lipid phases, colloidal materials and metal-organic frameworks is often characterized by small-angle X-ray scattering (SAXS). Frequently, residual disorder in these materials prevents high-resolution 3D structural characterization. Here we demonstrate that angular intensity variations in SAXS patterns can provide previously inaccessible information about local 3D structure via a rich, real-space distribution of three- and four-body statistics. We present the many-body characterisation of a monoolein-based hexagonal phase doped with a phospholipid, revealing non-uniform curvature in the lipid channels, likely due to phase separation of the lipids in the membrane. Our many-body technique has general applicability to nanomaterials with order in the range 10 nm−1 μm currently targeted by synchrotron SAXS and has the potential to impact diverse research areas within chemistry, biology and materials science. Less
Chemokines and their receptors mediate cell migration which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor chemokine recognition less is known about endogenous chemokine-induced receptor activation and G-protein coupling Here we present the cryo-electron microscopy structures of interleukin- IL- also known as CXCL -activated human CXC chemokine receptor CXCR in complex with Gi protein along with a crystal structure of CXCR bound to a designed allosteric antagonist Our results reveal a unique shallow mode of binding between CXCL and ... More
Chemokines and their receptors mediate cell migration, which influences multiple fundamental biological processes and disease conditions such as inflammation and cancer1. Although ample effort has been invested into the structural investigation of the chemokine receptors and receptor–chemokine recognition2,3,4, less is known about endogenous chemokine-induced receptor activation and G-protein coupling. Here we present the cryo-electron microscopy structures of interleukin-8 (IL-8, also known as CXCL8)-activated human CXC chemokine receptor 2 (CXCR2) in complex with Gi protein, along with a crystal structure of CXCR2 bound to a designed allosteric antagonist. Our results reveal a unique shallow mode of binding between CXCL8 and CXCR2, and also show the interactions between CXCR2 and Gi protein. Further structural analysis of the inactive and active states of CXCR2 reveals a distinct activation process and the competitive small-molecule antagonism of chemokine receptors. In addition, our results provide insights into how a G-protein-coupled receptor is activated by an endogenous protein molecule, which will assist in the rational development of therapeutics that target the chemokine system for better pharmacological profiles. Less
Of the eighteen hemagglutinin HA subtypes H H that have been identified in bats and aquatic birds many HA subtypes have been structurally characterized However several subtypes H H and H still require characterization To better understand all of these HA subtypes at the molecular level HA structures from an A H N A swine Missouri A an A H N A turkey Ontario an A H N A duck Memphis an A H N A mallard Gurjev and an A H N A wedge-tailed shearwater Western Australia were determined by X-ray crystallography at and resolution respectively The interactions between ... More
Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes (H8, H11 and H12) still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) (A/wedge-tailed shearwater/Western Australia/2576/1979 were determined by X-ray crystallography at 2.2Å, 2.3Å, 2.8Å, 3.0Å and 2.5Å resolution, respectively. The interactions between these viruses and host receptors were studied utilizing glycan-binding analyses with their recombinant HA. The data show that all avian HAs retain their strict binding preference to avian receptors, whereas swine H4 has a weak human receptor binding. The molecular characterization and structural analyses of the HA from these zoonotic influenza viruses not only provide a deeper appreciation and understanding of the structure of all HA subtypes, but also re-iterate why continuous global surveillance is needed. Less
The enzyme pyridoxal kinase PdxK catalyzes the conversion of pyridoxal to pyridoxal- -phosphate PLP using ATP as the co-factor The product pyridoxal- -phosphate plays a key role in several biological processes such as transamination decarboxylation and deamination In the present study full-length ORF of PdxK from Leishmania donovani LdPdxK was cloned and then purified using affinity chromatography LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH Biochemical analysis of LdPdxK with pyridoxal pyridoxamine pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK ... More
The enzyme pyridoxal kinase (PdxK) catalyzes the conversion of pyridoxal to pyridoxal-5′-phosphate (PLP) using ATP as the co-factor. The product pyridoxal-5′-phosphate plays a key role in several biological processes such as transamination, decarboxylation and deamination. In the present study, full-length ORF of PdxK from Leishmania donovani (LdPdxK) was cloned and then purified using affinity chromatography. LdPdxK exists as a homo-dimer in solution and shows more activity at near to physiological pH. Biochemical analysis of LdPdxK with pyridoxal, pyridoxamine, pyridoxine and ginkgotoxin revealed its affinity preference towards different substrates. The secondary structure analysis using circular dichroism spectroscopy showed LdPdxK to be predominantly α-helical in organization which tends to decline at lower and higher pH. Simultaneously, LdPdxK was crystallized and its three-dimensional structure in complex with ADP and different substrates were determined. Crystal structure of LdPdxK delineated that it has a central core of β-sheets surrounded by α-helices with a conserved GTGD ribokinase motif. The structures of LdPdxK disclosed no major structural changes between ADP and ADP- substrate bound structures. In addition, comparative structural analysis highlighted the key differences between the active site pockets of leishmanial and human PdxK, rendering LdPdxK an attractive candidate for the designing of novel and specific inhibitors. Less
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea They possess highly diverse biological functions Explosion of research on microbial rhodopsins led to breakthroughs in their applications in particular in neuroscience An unexpected new discovery was a Na -pumping KR rhodopsin from Krokinobacter eikastus the first light-driven non-proton cation pump A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and therefore Na pumping cannot be understood in the framework ... More
Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience. An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters. KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies. The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release. Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics. Less
Membrane-embedded sensor histidine kinases HKs and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment and are often crucial for their survival and pathogenicity The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms and regulate the activity of the cognate response regulator proteins with which they form two-component signaling systems TCSs Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed dubbed swinging piston helical rotation and diagonal scissoring Yet despite decades of studies there is no consensus on whether ... More
Membrane-embedded sensor histidine kinases (HKs) and chemoreceptors are used ubiquitously by bacteria and archaea to percept the environment, and are often crucial for their survival and pathogenicity. The proteins can transmit the signal from the sensor domain to the catalytic kinase domain reliably over the span of several hundreds of angstroms, and regulate the activity of the cognate response regulator proteins, with which they form two-component signaling systems (TCSs). Several mechanisms of transmembrane signal transduction in TCS receptors have been proposed, dubbed (swinging) piston, helical rotation, and diagonal scissoring. Yet, despite decades of studies, there is no consensus on whether these mechanisms are common for all TCS receptors. Here, we extend our previous work on Escherichia coli nitrate/nitrite sensor kinase NarQ. We determined a crystallographic structure of the sensor-TM-HAMP fragment of the R50S mutant, which, unexpectedly, was found in a ligand-bound-like conformation, despite an inability to bind nitrate. Subsequently, we reanalyzed the structures of the ligand-free and ligand-bound NarQ and NarX sensor domains, and conducted extensive molecular dynamics simulations of ligand-free and ligand-bound wild type and mutated NarQ. Based on the data, we show that binding of nitrate to NarQ causes, first and foremost, helical rotation and diagonal scissoring of the α-helices at the core of the sensor domain. These conformational changes are accompanied by a subtle piston-like motion, which is amplified by a switch in the secondary structure of the linker between the sensor and TM domains. We conclude that helical rotation, diagonal scissoring, and piston are simply different degrees of freedom in coiled-coil proteins and are not mutually exclusive in NarQ, and likely in other nitrate sensors and TCS proteins as well. Less
Light-oxygen-voltage LOV domains are ubiquitous photosensory modules found in proteins from bacteria archaea and eukaryotes Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics with improved versions being continuously developed Many of the engineering efforts focused on the thermal stabilization of LOV domains Recently we described a naturally thermostable LOV domain from Chloroflexus aggregans Here we show that the discovered protein can be further stabilized using proline substitution We tested the effects of three mutations and found that the melting temperature of the A P mutant is raised by approximately C whereas mutations A P ... More
Light-oxygen-voltage (LOV) domains are ubiquitous photosensory modules found in proteins from bacteria, archaea and eukaryotes. Engineered versions of LOV domains have found widespread use in fluorescence microscopy and optogenetics, with improved versions being continuously developed. Many of the engineering efforts focused on the thermal stabilization of LOV domains. Recently, we described a naturally thermostable LOV domain from Chloroflexus aggregans. Here we show that the discovered protein can be further stabilized using proline substitution. We tested the effects of three mutations, and found that the melting temperature of the A95P mutant is raised by approximately 2 °C, whereas mutations A56P and A58P are neutral. To further evaluate the effects of mutations, we crystallized the variants A56P and A95P, while the variant A58P did not crystallize. The obtained crystal structures do not reveal any alterations in the proteins other than the introduced mutations. Molecular dynamics simulations showed that mutation A58P alters the structure of the respective loop (Aβ-Bβ), but does not change the general structure of the protein. We conclude that proline substitution is a viable strategy for the stabilization of the Chloroflexus aggregans LOV domain. Since the sequences and structures of the LOV domains are overall well-conserved, the effects of the reported mutations may be transferable to other proteins belonging to this family. Less
Perovskites have seen significant research interest in the last decade As ternary and quaternary compounds their chemical space is exceptionally large yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error Here we report a high-throughput experimental framework for the discovery of new perovskite single crystals We use machine learning ML to guide the sequence of ever-improved robotic synthetic trials We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition We then use an ML ... More
Perovskites have seen significant research interest in the last decade. As ternary and quaternary compounds, their chemical space is exceptionally large, yet perovskite development has been limited to a restricted set of chemical constituents often discovered through trial and error. Here, we report a high-throughput experimental framework for the discovery of new perovskite single crystals. We use machine learning (ML) to guide the sequence of ever-improved robotic synthetic trials. We perform high-throughput syntheses of perovskite single crystals with a protein crystallization robot and characterize the outcomes with the aid of convolutional neural network-based image recognition. We then use an ML model to predict the optimal conditions for the synthesis of a new perovskite single crystal, enabling us to report the first synthesis of (3-PLA)2PbCl4.This material exhibits strong blue emission, illustrating the applicability of the method in identifying new optoelectronic materials. Less
Glucagon-like peptide- receptor GLP- R is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type diabetes Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides however little is known about their extracellular domain ECD conformations in the absence of orthosteric ligands which has limited our understanding of their activation mechanism Here we report the resolution peptide-free crystal structure of the full-length human GLP- R in an inactive state which reveals a unique closed conformation of the ECD Disulfide cross-linking validates the physiological relevance of the ... More
Glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein-coupled receptor that plays an important role in glucose homeostasis and treatment of type 2 diabetes. Structures of full-length class B receptors were determined in complex with their orthosteric agonist peptides, however, little is known about their extracellular domain (ECD) conformations in the absence of orthosteric ligands, which has limited our understanding of their activation mechanism. Here, we report the 3.2 Å resolution, peptide-free crystal structure of the full-length human GLP-1R in an inactive state, which reveals a unique closed conformation of the ECD. Disulfide cross-linking validates the physiological relevance of the closed conformation, while electron microscopy (EM) and molecular dynamic (MD) simulations suggest a large degree of conformational dynamics of ECD that is necessary for binding GLP-1. Our inactive structure represents a snapshot of the peptide-free GLP-1R and provides insights into the activation pathway of this receptor family. Less
Two-component signaling systems TCSs are a large and important class of sensory systems in bacteria archaea and some eukaryotes yet their mechanism of action is still not fully understood from the structural point of view Many TCS receptors are elongated flexible proteins with transmembrane TM regions and are difficult to work with Consequently truncated fragments of the receptors are often used in structural studies However it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins Recently we crystallized a fragment of Escherichia coli nitrate nitrite sensor histidine ... More
Two-component signaling systems (TCSs) are a large and important class of sensory systems in bacteria, archaea, and some eukaryotes, yet their mechanism of action is still not fully understood from the structural point of view. Many TCS receptors are elongated flexible proteins with transmembrane (TM) regions, and are difficult to work with. Consequently, truncated fragments of the receptors are often used in structural studies. However, it is not fully clear whether the structures of the fragments correspond well to their native structures in the context of full-length proteins. Recently, we crystallized a fragment of Escherichia coli nitrate/nitrite sensor histidine kinase, NarQ, encompassing the sensor, TM, and HAMP domains. Here we report that a smaller proteolytic fragment consisting of the sensor and TM domains can also be crystallized using the in meso approach. The structure of the fragment is similar to the previously determined one, with minor differences in the vicinity of the truncation site. The results show that the crystallization of such sensor–TM fragments can be accomplished and can provide information on the packing of transmembrane helices, albeit limited, and that the proteolysis may or may not be a problem during crystallization. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs has recently been discovered Unlike in the known rhodopsins in HeRs the N termini face the cytoplasm The function of HeRs remains unknown We present the structures of the bacterial HeR- C in two states at the resolution of which highlight its remarkable difference from all known rhodopsins The interior of HeR s extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity Schiff base cavity SBC surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR’s extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes. Less
Human muscarinic receptor M belongs to the class A subfamily of the G-protein-coupled receptors GPCRs M has emerged as an attractive drug target for the treatment of Alzheimer s disease and schizophrenia Recent results showed that M -mediated cholinergic transmission is related to motor symptoms in Parkinson s disease Selective ligand design for the five muscarinic acetylcholine receptor mAchR subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets In order to obtain M -selective antagonists a new approach was tried to lock M into an inactive form by rationally designing an N ... More
Human muscarinic receptor M4 belongs to the class A subfamily of the G-protein-coupled receptors (GPCRs). M4 has emerged as an attractive drug target for the treatment of Alzheimer’s disease and schizophrenia. Recent results showed that M4-mediated cholinergic transmission is related to motor symptoms in Parkinson’s disease. Selective ligand design for the five muscarinic acetylcholine receptor (mAchR) subtypes currently remains challenging owing to the high sequence and structural similarity of their orthosteric binding pockets. In order to obtain M4-selective antagonists, a new approach was tried to lock M4 into an inactive form by rationally designing an N4497.49R mutation, which mimics the allosteric sodium binding in the conserved sodium site usually found in class A GPCRs. In addition, the crystal structure of the mutation-induced inactive M4 was determined. By comparative analysis with other mAchR structures, followed by functional assays, the N4497.49R mutation was shown to stabilize M4 into an inactive state. Virtual screening of a focused ligand library using the crystal structure showed that the inactive M4 prefers antagonists much more than agonists. This study provides a powerful mutation strategy to stabilize GPCRs in inactive states and facilitate their structure determination. Less
GPR is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington s disease and several psychiatric disorders Pathological malfunction of GPR signalling occurs primarily through the heterotrimeric Gs protein but it is unclear how GPR and Gs couple for signal transduction and whether a native ligand or other activating input is required Here we present the high-resolution structures of human GPR in three states a ligand-free state a Gs-coupled self-activation state and a potential allosteric ligand-bound state Together our structures reveal that extracellular loop occupies the ... More
GPR52 is a class-A orphan G-protein-coupled receptor that is highly expressed in the brain and represents a promising therapeutic target for the treatment of Huntington’s disease and several psychiatric disorders1,2. Pathological malfunction of GPR52 signalling occurs primarily through the heterotrimeric Gs protein2, but it is unclear how GPR52 and Gs couple for signal transduction and whether a native ligand or other activating input is required. Here we present the high-resolution structures of human GPR52 in three states: a ligand-free state, a Gs-coupled self-activation state and a potential allosteric ligand-bound state. Together, our structures reveal that extracellular loop 2 occupies the orthosteric binding pocket and operates as a built-in agonist, conferring an intrinsically high level of basal activity to GPR523. A fully active state is achieved when Gs is coupled to GPR52 in the absence of an external agonist. The receptor also features a side pocket for ligand binding. These insights into the structure and function of GPR52 could improve our understanding of other self-activated GPCRs, enable the identification of endogenous and tool ligands, and guide drug discovery efforts that target GPR52. Less
Phospholipase D enzymes PLDs are ubiquitous phosphodiesterases that produce phosphatidic acid PA a key second messenger and biosynthetic building block Although an orthologous bacterial Streptomyces sp strain PMF PLD structure was solved two decades ago the molecular basis underlying the functions of the human PLD enzymes hPLD remained unclear based on this structure due to the low homology between these sequences Here we describe the first crystal structures of hPLD and hPLD catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes Furthermore structure-based mutation studies and structures of inhibitor hPLD complexes allowed us ... More
Phospholipase D enzymes (PLDs) are ubiquitous phosphodiesterases that produce phosphatidic acid (PA), a key second messenger and biosynthetic building block. Although an orthologous bacterial Streptomyces sp. strain PMF PLD structure was solved two decades ago, the molecular basis underlying the functions of the human PLD enzymes (hPLD) remained unclear based on this structure due to the low homology between these sequences. Here, we describe the first crystal structures of hPLD1 and hPLD2 catalytic domains and identify novel structural elements and functional differences between the prokaryotic and eukaryotic enzymes. Furthermore, structure-based mutation studies and structures of inhibitor–hPLD complexes allowed us to elucidate the binding modes of dual and isoform-selective inhibitors, highlight key determinants of isoenzyme selectivity and provide a basis for further structure-based drug discovery and functional characterization of this therapeutically important superfamily of enzymes. Less
Rhodopsins are the most abundant light-harvesting proteins A new family of rhodopsins heliorhodopsins HeRs was recently discovered In opposite to the known rhodopsins their N-termini face the cytoplasm HeRs structure and function remain unknown We present structures of two HeR- C states at showing its remarkable difference from all known rhodopsins Its internal extracellular part is completely hydrophobic while the cytoplasmic part comprises a cavity active site surrounded by charged amino acids and containing a cluster of water molecules presumably being a primary proton acceptor from the Schiff base At acidic pH a planar triangle molecule acetate is present in ... More
Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH a planar triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement into fundamental redox biological processes. Less
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB and CB Their high sequence similarity low agonist selectivity and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications Importantly missing structural information has significantly held back the development of promising CB -selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB Here we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB and CB in complex with Gi as well as agonist-bound CB crystal structure Of important scientific and therapeutic benefit our results reveal a ... More
Human endocannabinoid systems modulate multiple physiological processes mainly through the activation of cannabinoid receptors CB1 and CB2. Their high sequence similarity, low agonist selectivity, and lack of activation and G protein-coupling knowledge have hindered the development of therapeutic applications. Importantly, missing structural information has significantly held back the development of promising CB2-selective agonist drugs for treating inflammatory and neuropathic pain without the psychoactivity of CB1. Here, we report the cryoelectron microscopy structures of synthetic cannabinoid-bound CB2 and CB1 in complex with Gi, as well as agonist-bound CB2 crystal structure. Of important scientific and therapeutic benefit, our results reveal a diverse activation and signaling mechanism, the structural basis of CB2-selective agonists design, and the unexpected interaction of cholesterol with CB1, suggestive of its endogenous allosteric modulating role. Less
Shiga toxin Stx is the major virulence factor of Shiga toxin-producing Escherichia coli STEC Stx evolves rapidly and as such new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies A new subtype Stx k was recently identified in E coli isolated from a wide range of sources including diarrheal patients animals and raw meats and was poorly detected by existing immunoassays In this study the structure of Stx kE Q was determined at resolution and the conservation of structure with Stx a was revealed A novel polyclonal antibody capable of neutralizing Stx k ... More
Shiga toxin (Stx) is the major virulence factor of Shiga toxin-producing Escherichia coli (STEC). Stx evolves rapidly and, as such, new subtypes continue to emerge that challenge the efficacy of existing disease management and surveillance strategies. A new subtype, Stx2k, was recently identified in E. coli isolated from a wide range of sources including diarrheal patients, animals, and raw meats, and was poorly detected by existing immunoassays. In this study, the structure of Stx2kE167Q was determined at 2.29 Å resolution and the conservation of structure with Stx2a was revealed. A novel polyclonal antibody capable of neutralizing Stx2k and an immunoassay, with a 10-fold increase in sensitivity compared to assays using extant antibodies, were developed. Stx2k is less toxic than Stx2a in Vero cell assays but is similar to Stx2a in receptor-binding preference, thermostability, and acid tolerance. Although Stx2k does not appear to be as potent as Stx2a to Vero cells, the wide distribution and blended virulence profiles of the Stx2k-producing strains suggest that horizontal gene transfer through Stx2k-converting phages could result in the emergence of new and highly virulent pathogens. This study provides useful information and tools for early detection and control of Stx2k-producing E. coli, which could reduce public risk of infection by less-known STECs. Less
Membrane integral ATP synthases produce adenosine triphosphate the universal energy currency of most organisms However important details of proton driven energy conversion are still unknown We present the first high-resolution structure of the in meso crystallized c-ring of subunits from spinach chloroplasts The structure reveals molecular mechanisms of intersubunit contacts in the c -ring and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane Similar densities were found in all known high-resolution structures of c-rings of F FO ATP synthases from archaea and bacteria to eukaryotes The densities might originate from isoprenoid quinones ... More
Membrane integral ATP synthases produce adenosine triphosphate, the universal “energy currency” of most organisms. However, important details of proton driven energy conversion are still unknown. We present the first high-resolution structure (2.3 Å) of the in meso crystallized c-ring of 14 subunits from spinach chloroplasts. The structure reveals molecular mechanisms of intersubunit contacts in the c14-ring, and it shows additional electron densities inside the c-ring which form circles parallel to the membrane plane. Similar densities were found in all known high-resolution structures of c-rings of F1FO ATP synthases from archaea and bacteria to eukaryotes. The densities might originate from isoprenoid quinones (such as coenzyme Q in mitochondria and plastoquinone in chloroplasts) that is consistent with differential UV-Vis spectroscopy of the c-ring samples, unusually large distance between polar/apolar interfaces inside the c-ring and universality among different species. Although additional experiments are required to verify this hypothesis, coenzyme Q and its analogues known as electron carriers of bioenergetic chains may be universal cofactors of ATP synthases, stabilizing c-ring and prevent ion leakage through it. Less
Cysteinyl leukotriene G protein-coupled receptors CysLT and CysLT regulate pro-inflammatory responses associated with allergic disorders While selective inhibition of CysLT R has been used for treating asthma and associated diseases for over two decades CysLT R has recently started to emerge as a potential drug target against atopic asthma brain injury and central nervous system disorders as well as several types of cancer Here we describe four crystal structures of CysLT R in complex with three dual CysLT R CysLT R antagonists The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular ... More
Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT2R in complex with three dual CysLT1R/CysLT2R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants. Less
Broadly HIV- neutralizing VRC class antibodies target the CD -binding site of Env They are derived from VH - antibody heavy chains paired with rare light chains expressing -amino acid-long CDRL s They have been isolated from infected subjects but have not yet been elicited by immunization Env-derived immunogens capable of binding the germline forms of VRC B cell receptors on naive B cells have been designed and evaluated in knockin mice However the elicited antibodies cannot bypass glycans present on the conserved position N of Env which restricts access to the CD -binding site Efforts to guide the appropriate ... More
Broadly HIV-1 neutralizing VRC01 class antibodies target the CD4-binding site of Env. They are derived from VH1-2*02 antibody heavy chains paired with rare light chains expressing 5-amino acid-long CDRL3s. They have been isolated from infected subjects but have not yet been elicited by immunization. Env-derived immunogens capable of binding the germline forms of VRC01 B cell receptors on naive B cells have been designed and evaluated in knockin mice. However, the elicited antibodies cannot bypass glycans present on the conserved position N276 of Env, which restricts access to the CD4-binding site. Efforts to guide the appropriate maturation of these antibodies by sequential immunization have not yet been successful. Here, we report on a two-step immunization scheme that leads to the maturation of VRC01-like antibodies capable of accommodating the N276 glycan and displaying autologous tier 2 neutralizing activities. Our results are relevant to clinical trials aiming to elicit VRC01 antibodies. Less
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding and understanding this diversity is key for designing selective or biased drugs for them Here we determine two crystal structures of the a A adrenergic receptor a AAR in complex with a partial agonist and an antagonist Key non-conserved residues from the ligand-binding pocket Phe and Tyr to G-protein coupling region Ile and Lys are discovered to play a key role in the interplay between partial agonism and biased signaling of a AAR which provides insights into the diversity of ligand ... More
Adrenergic receptors are highly homologous while at the same time display a wide diversity of ligand and G-protein binding, and understanding this diversity is key for designing selective or biased drugs for them. Here, we determine two crystal structures of the a2A adrenergic receptor (a2AAR) in complex with a partial agonist and an antagonist. Key non-conserved residues from the ligand-binding pocket (Phe7.39 and Tyr6.55) to G-protein coupling region (Ile34.51 and Lys34.56) are discovered to play a key role in the interplay between partial agonism and biased signaling of a2AAR, which provides insights into the diversity of ligand binding and G-protein coupling preference of adrenergic receptors and lays the foundation for the discovery of next-generation drugs targeting these receptors. Less
Adrenergic G-protein-coupled receptors GPCRs mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions Extensive studies have been carried out to investigate the structure and function of adrenergic receptors ARs However the structure of a adrenergic receptors aARs remains to be determined Here we report the structure of the human a C adrenergic receptor a CAR with the non-selective antagonist RS at Our structure mutations modeling and functional experiments indicate that a a CAR-specific D ECL -R ECL -Y network plays a role in determining a adrenergic subtype selectivity ... More
Adrenergic G-protein-coupled receptors (GPCRs) mediate different cellular signaling pathways in the presence of endogenous catecholamines and play important roles in both physiological and pathological conditions. Extensive studies have been carried out to investigate the structure and function of � adrenergic receptors (�ARs). However, the structure of a adrenergic receptors (aARs) remains to be determined. Here, we report the structure of the human a2C adrenergic receptor (a2CAR) with the non-selective antagonist, RS79948, at 2.8 �. Our structure, mutations, modeling, and functional experiments indicate that a a2CAR-specific D206ECL2-R409ECL3-Y4056.58 network plays a role in determining a2 adrenergic subtype selectivity. Furthermore, our results show that a specific loosened helix at the top of TM4 in a2CAR is involved in receptor activation. Together, our structure of human a2CAR-RS79948 provides key insight into the mechanism underlying the a2 adrenergic receptor activation and subtype selectivity. Less