1194 Citations
The human neuropeptide Y NPY Y receptor Y R plays essential roles in food intake bone formation and mood regulation and has been considered an important drug target for obesity and anxiety However development of drugs targeting Y R remains challenging with no success in clinical application yet Here we report the crystal structure of Y R bound to a selective antagonist JNJ- at resolution The structure reveals molecular details of the ligand-binding mode of Y R Combined with mutagenesis studies the Y R structure provides insights into key factors that define antagonistic activity of diverse antagonists Comparison with the ... More
The human neuropeptide Y (NPY) Y2 receptor (Y2R) plays essential roles in food intake, bone formation and mood regulation, and has been considered an important drug target for obesity and anxiety. However, development of drugs targeting Y2R remains challenging with no success in clinical application yet. Here, we report the crystal structure of Y2R bound to a selective antagonist JNJ-31020028 at 2.8 Å resolution. The structure reveals molecular details of the ligand-binding mode of Y2R. Combined with mutagenesis studies, the Y2R structure provides insights into key factors that define antagonistic activity of diverse antagonists. Comparison with the previously determined antagonist-bound Y1R structures identified receptor-ligand interactions that play different roles in modulating receptor activation and mediating ligand selectivity. These findings deepen our understanding about molecular mechanisms of ligand recognition and subtype specificity of NPY receptors, and would enable structure-based drug design. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications. It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects. Fully automated data collection is now possible at several synchrotrons, which means that the number of samples tested at the synchrotron is currently increasing rapidly. Therefore, the protein crystallography research communities at the University of Oulu, Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron. In IceBear, each crystal selected for data collection is given a unique sample name and a crystal page is generated. Subsequently, the metadata required for data collection are uploaded directly to the ISPyB synchrotron database by a shipment module, and for each sample a link to the relevant ISPyB page is stored. IceBear allows notes to be made for each sample during cryocooling treatment and during data collection, as well as in later steps of the structure determination. Protocols are also available to aid the recycling of pins, pucks and dewars when the dewar returns from the synchrotron. The IceBear database is organized around projects, and project members can easily access the crystallization and diffraction metadata for each sample, as well as any additional information that has been provided via the notes. The crystal page for each sample connects the crystallization, diffraction and structural information by providing links to the IceBear drop-viewer page and to the ISPyB data-collection page, as well as to the structure deposited in the Protein Data Bank. Less
The Parkin co-regulated gene protein PACRG binds at the inner junction between doublet microtubules of the axoneme a structure found in flagella and cilia PACRG binds to the adaptor protein meiosis expressed gene MEIG but how they bind to microtubules is unknown Here we report the crystal structure of human PACRG in complex with MEIG PACRG adopts a helical repeat fold with a loop that interacts with MEIG Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation ... More
The Parkin co-regulated gene protein (PACRG) binds at the inner junction between doublet microtubules of the axoneme, a structure found in flagella and cilia. PACRG binds to the adaptor protein meiosis expressed gene 1 (MEIG1), but how they bind to microtubules is unknown. Here, we report the crystal structure of human PACRG in complex with MEIG1. PACRG adopts a helical repeat fold with a loop that interacts with MEIG1. Using the structure of the axonemal doublet microtubule from the protozoan Chlamydomonas reinhardtii and single-molecule fluorescence microscopy, we propose that PACRG binds to microtubules while simultaneously recruiting free tubulin to catalyze formation of the inner junction. We show that the homologous PACRG-like protein also mediates dual tubulin interactions but does not bind MEIG1. Our findings establish a framework to assess the function of the PACRG family of proteins and MEIG1 in regulating axoneme assembly. Less
Mutations in two different domains of the ubiquitously expressed TRIM protein give rise to two clinically separate diseases one of which is Limb-girdle muscular dystrophy type H LGMD H Uncovering the muscle-specific role of TRIM in LGMD H pathogenesis has proven difficult as neurogenic phenotypes independent of LGMD H pathology are present in TRIM KO mice We previously established a platform to study LGMD H pathogenesis using Drosophila melanogaster as a model Here we show that LGMD H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM Furthermore transgenic expression of a subset ... More
Mutations in two different domains of the ubiquitously expressed TRIM32 protein give rise to two clinically separate diseases, one of which is Limb-girdle muscular dystrophy type 2H (LGMD2H). Uncovering the muscle-specific role of TRIM32 in LGMD2H pathogenesis has proven difficult, as neurogenic phenotypes, independent of LGMD2H pathology, are present in TRIM32 KO mice. We previously established a platform to study LGMD2H pathogenesis using Drosophila melanogaster as a model. Here we show that LGMD2H disease-causing mutations in the NHL domain are molecularly and structurally conserved between fly and human TRIM32. Furthermore, transgenic expression of a subset of myopathic alleles (R394H, D487N, and 520fs) induce myofibril abnormalities, altered nuclear morphology, and reduced TRIM32 protein levels, mimicking phenotypes in patients afflicted with LGMD2H. Intriguingly, we also report for the first time that the protein levels of βPS integrin and sarcoglycan δ, both core components of costameres, are elevated in TRIM32 disease-causing alleles. Similarly, murine myoblasts overexpressing a catalytically inactive TRIM32 mutant aberrantly accumulate α- and β-dystroglycan and α-sarcoglycan. We speculate that the stoichiometric loss of costamere components disrupts costamere complexes to promote muscle degeneration. Less
The web-based IceBear software is a versatile tool to monitor the results of crystallization experiments and is designed to facilitate supervisor and student communications It also records and tracks all relevant information from crystallization setup to PDB deposition in protein crystallography projects Fully automated data collection is now possible at several synchrotrons which means that the number of samples tested at the synchrotron is currently increasing rapidly Therefore the protein crystallography research communities at the University of Oulu Weizmann Institute of Science and Diamond Light Source have joined forces to automate the uploading of sample metadata to the synchrotron In ... More
The web-based IceBear software is a versatile tool to monitor the results of
crystallization experiments and is designed to facilitate supervisor and student
communications. It also records and tracks all relevant information from
crystallization setup to PDB deposition in protein crystallography projects. Fully
automated data collection is now possible at several synchrotrons, which means
that the number of samples tested at the synchrotron is currently increasing
rapidly. Therefore, the protein crystallography research communities at the
University of Oulu, Weizmann Institute of Science and Diamond Light Source
have joined forces to automate the uploading of sample metadata to the
synchrotron. In IceBear, each crystal selected for data collection is given a
unique sample name and a crystal page is generated. Subsequently, the metadata
required for data collection are uploaded directly to the ISPyB synchrotron
database by a shipment module, and for each sample a link to the relevant
ISPyB page is stored. IceBear allows notes to be made for each sample during
cryocooling treatment and during data collection, as well as in later steps of the
structure determination. Protocols are also available to aid the recycling of pins,
pucks and dewars when the dewar returns from the synchrotron. The IceBear
database is organized around projects, and project members can easily access
the crystallization and diffraction metadata for each sample, as well as any
additional information that has been provided via the notes. The crystal page for
each sample connects the crystallization, diffraction and structural information
by providing links to the IceBear drop-viewer page and to the ISPyB datacollection page, as well as to the structure deposited in the Protein Data Bank. Less
crystallization experiments and is designed to facilitate supervisor and student
communications. It also records and tracks all relevant information from
crystallization setup to PDB deposition in protein crystallography projects. Fully
automated data collection is now possible at several synchrotrons, which means
that the number of samples tested at the synchrotron is currently increasing
rapidly. Therefore, the protein crystallography research communities at the
University of Oulu, Weizmann Institute of Science and Diamond Light Source
have joined forces to automate the uploading of sample metadata to the
synchrotron. In IceBear, each crystal selected for data collection is given a
unique sample name and a crystal page is generated. Subsequently, the metadata
required for data collection are uploaded directly to the ISPyB synchrotron
database by a shipment module, and for each sample a link to the relevant
ISPyB page is stored. IceBear allows notes to be made for each sample during
cryocooling treatment and during data collection, as well as in later steps of the
structure determination. Protocols are also available to aid the recycling of pins,
pucks and dewars when the dewar returns from the synchrotron. The IceBear
database is organized around projects, and project members can easily access
the crystallization and diffraction metadata for each sample, as well as any
additional information that has been provided via the notes. The crystal page for
each sample connects the crystallization, diffraction and structural information
by providing links to the IceBear drop-viewer page and to the ISPyB datacollection page, as well as to the structure deposited in the Protein Data Bank. Less
The unique crystallization properties of the antenna protein C-phycocyanin C-PC from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives Remarkably the crystal dimensions vary from a few micrometres as used in serial crystallography to several hundred micrometres with a very diverse crystal morphology More than unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed The addition of small-molecule additives revealed three new crystal packings of C-PC which are discussed ... More
The unique crystallization properties of the antenna protein C-phycocyanin (C-PC) from the thermophilic cyanobacterium Thermosynechococcus elongatus are reported and discussed. C-PC crystallizes in hundreds of significantly different conditions within a broad pH range and in the presence of a wide variety of precipitants and additives. Remarkably, the crystal dimensions vary from a few micrometres, as used in serial crystallography, to several hundred micrometres, with a very diverse crystal morphology. More than 100 unique single-crystal X-ray diffraction data sets were collected from randomly selected crystals and analysed. The addition of small-molecule additives revealed three new crystal packings of C-PC, which are discussed in detail. The high propensity of this protein to crystallize, combined with its natural blue colour and its fluorescence characteristics, make it an excellent candidate as a superior and highly adaptable model system in crystallography. C-PC can be used in technical and methods development approaches for X-ray and neutron diffraction techniques, and as a system for comprehending the fundamental principles of protein crystallography. 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
Myxococcus xanthus displays two types of motilities i e Social S and Adventurous A The pole-to-pole reversals of these motility regulator proteins is the key to this process Here we determined resolution crystal structure of MglC which revealed that despite sharing sequence identity both MglB and MglC adopt Regulatory Light Chain RLC family fold Interestingly MglC is structurally unique compared to the other known RLC family proteins having - shift in the orientation of functionally important helix Using isothermal titration calorimetry and gel filtration chromatography we show that MglC binds MglB in stoichiometry with submicromolar range dissociation constant Using combination ... More
Myxococcus xanthus displays two types of motilities i.e. Social (S) and Adventurous (A). The pole-to-pole reversals of these motility regulator proteins is the key to this process. Here, we determined ~1.85 Å resolution crystal structure of MglC, which revealed that despite sharing <9% sequence identity, both MglB and MglC adopt Regulatory Light Chain 7 (RLC7) family fold. Interestingly, MglC is structurally unique compared to the other known RLC7 family proteins having ~30°-40° shift in the orientation of functionally important α2 helix. Using isothermal titration calorimetry and gel filtration chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using combination of small angle X-ray scattering and molecular docking studies, we show that MglBC complex is formed by MglC homodimer sandwiched between two homodimers of MglB. Less
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges Hydrogel microsphere formulations offer a route to quicker patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force Crystals of an intact monoclonal antibody are prepared as a concentrated suspension mg mL which is then encapsulated within hydrogel microspheres with diameters as small as m The hydrogel microspheres ... More
Monoclonal antibodies are therapeutic molecules known for their high specificity and versatility in the treatment of cancer and autoimmune disorders, but dosage forms are typically limited to low concentrations and large fluid volumes due to formulation challenges. Hydrogel microsphere formulations offer a route to quicker, patient-friendly dosing regimens for monoclonal antibodies with high loading and favorable flow properties needed for injection through a narrow syringe needle under moderate applied force. Crystals of an intact monoclonal antibody are prepared as a concentrated suspension (>300 mg mL−1) which is then encapsulated within hydrogel microspheres with diameters as small as 30 µm. The hydrogel microspheres contain up to 56 wt% (dry basis) monoclonal antibody and release within 4 days under in vitro dissolution conditions. The hydrogel microspheres are concentrated into densely packed suspensions containing up to 300 mg mL−1 monoclonal antibody to evaluate their flow. These hydrogel formulations shear-thin and have lower viscosity when compared to both liquid and suspended crystal forms of the monoclonal antibody, demonstrating the potential of hydrogel microsphere encapsulants as a carrier which can mask undesirable flow properties of concentrated antibody therapeutics. 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
Neurotensin receptor NTSR and related G protein coupled receptors of the ghrelin family are clinically unexploited and several mechanistic aspects of their activation and inactivation have remained unclear Enabled by a new crystallization design we present five new structures apo-state NTSR as well as complexes with nonpeptide inverse agonists SR and SR A partial agonist RTI- a and the novel full agonist SRI- providing structural rationales on how ligands modulate NTSR The inverse agonists favor a large extracellular opening of helices VI and VII undescribed so far for NTSR causing a constriction of the intracellular portion In contrast the full ... More
Neurotensin receptor 1 (NTSR1) and related G protein–coupled receptors of the ghrelin family are clinically unexploited, and several mechanistic aspects of their activation and inactivation have remained unclear. Enabled by a new crystallization design, we present five new structures: apo-state NTSR1 as well as complexes with nonpeptide inverse agonists SR48692 and SR142948A, partial agonist RTI-3a, and the novel full agonist SRI-9829, providing structural rationales on how ligands modulate NTSR1. The inverse agonists favor a large extracellular opening of helices VI and VII, undescribed so far for NTSR1, causing a constriction of the intracellular portion. In contrast, the full and partial agonists induce a binding site contraction, and their efficacy correlates with the ability to mimic the binding mode of the endogenous agonist neurotensin. Providing evidence of helical and side-chain rearrangements modulating receptor activation, our structural and functional data expand the mechanistic understanding of NTSR1 and potentially other peptidergic receptors. Less
Background Huntington's disease HD is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors Using shotgun sequencing we investigated the gut microbiome composition in the R transgenic mouse model of HD from to weeks of age early adolescent through to adult stages Targeted metabolomics was also performed on the blood plasma of these mice n per group at weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile Results ... More
Background Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder with onset and severity of symptoms influenced by various environmental factors. Recent discoveries have highlighted the importance of the gastrointestinal microbiome in mediating the gut-brain-axis bidirectional communication via circulating factors. Using shotgun sequencing, we investigated the gut microbiome composition in the R6/1 transgenic mouse model of HD from 4 to 12 weeks of age (early adolescent through to adult stages). Targeted metabolomics was also performed on the blood plasma of these mice (n = 9 per group) at 12 weeks of age to investigate potential effects of gut dysbiosis on the plasma metabolome profile. Results Modelled time profiles of each species, KEGG Orthologs and bacterial genes, revealed heightened volatility in the R6/1 mice, indicating potential early effects of the HD mutation in the gut. In addition to gut dysbiosis in R6/1 mice at 12 weeks of age, gut microbiome function was perturbed. In particular, the butanoate metabolism pathway was elevated, suggesting increased production of the protective SCFA, butyrate, in the gut. No significant alterations were found in the plasma butyrate and propionate levels in the R6/1 mice at 12 weeks of age. The statistical integration of the metagenomics and metabolomics unraveled several Bacteroides species that were negatively correlated with ATP and pipecolic acid in the plasma. Conclusions The present study revealed the instability of the HD gut microbiome during the pre-motor symptomatic stage of the disease which may have dire consequences on the host's health. Perturbation of the HD gut microbiome function prior to significant cognitive and motor dysfunction suggest the potential role of the gut in modulating the pathogenesis of HD, potentially via specific altered plasma metabolites which mediate gut-brain signaling. 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
In this thesis the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin NmHR is presented Members of the rhodopsin family such as NmHR are integral membrane proteins comprising seven helices and a retinal chromophore which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive Through photoactivation the retinal chromophore undergoes an isomerization reaction which then drives conformational changes in the protein Through variations in residue composition rhodopsin can catalyze diverse chemical reactions including pumping protons sodium or chloride ions Chloride transport is a fundamental process in biology and crucial ... More
In this thesis, the chloride transport mechanism of the light-driven microbial chloride pump Nonlabens marinus halorhodopsin (NmHR) is presented. Members of the rhodopsin family, such as NmHR, are integral membrane proteins comprising seven α helices and a retinal chromophore, which is covalently bound to the protein via a protonated Schiff base and renders the proteins photoactive. Through photoactivation, the retinal chromophore undergoes an isomerization reaction, which then drives conformational changes in the protein. Through variations in residue composition, rhodopsin can catalyze diverse chemical reactions, including pumping protons, sodium, or chloride ions. Chloride transport is a fundamental process in biology and crucial for maintaining the electrochemical balance of the cell.
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
The advent of bright X-ray light sources such as third-generation synchrotrons and X ray free electron lasers has resulted in the emergence of time-resolved serial crystallography. These novel serial crystallography methods were combined with time resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations to study conformational changes and chloride translocation in NmHR after photoactivation. Five active state structural intermediates, determined in the picosecond to microsecond time domain, have been determined at the X-ray free electron laser. Structural insight into the late photocycle of NmHR was provided by time-resolved serial crystallography at the synchrotron, resulting in ten additional active state intermediates in the millisecond time domain. In addition, a new method was developed that allowed tracing of the anomalous substructure in the photostationary state, providing critical clues on the anion transport pathway in NmHR.
Together with resolving the position of four new transient chloride binding sites in time, the mechanism driving chloride transport is proposed based on the observed conformational changes of the protein after photoactivation. In summary, in the resting state chloride interacts with the protonated Schiff base of the retinal chromophore. Upon absorption of a photon, the retinal chromophore then isomerizes from the all trans to 13-cis configuration, which flips the protonated Schiff base and disrupts the interaction with the chloride ion. In the following step, the chloride translocation is initiated as the anion is pulled over the retinal chromophore to reestablish the interaction with the positive charge on the protonated Schiff base. After chloride is released into the exit tunnel to further diffuse towards the cytoplasm, a steric gate prevents chloride from flowing back into the dark state binding site. At the same time as the release of the chloride ion into the cytoplasm, a new anion is taken up from the extracellular space. In the uptake tunnel, the anion encounters a bottleneck formed by a salt bridge between an arginine and aspartate residue which forms an electrostatic gate. Upon opening of this electrostatic gate, chloride can enter the retinal binding pocket, a hydrophilic cavity in which the dark state binding site is located. Together with the closure of the electrostatic gate, the retinal chromophore isomerizes back to the all-trans-configuration, and the dark state chloride binding site is regenerated.
This thesis thereby presents the first detailed structural dynamics of ion transport by a chloride pumping rhodopsin and demonstrates the capabilities of novel serial crystallography methods. Less
How have complex brains evolved from simple circuits Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei the output structures of the cerebellum Using single-nucleus RNA sequencing in mice chickens and humans as well as STARmap spatial transcriptomic analysis and whole central nervous system projection tracing we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in ... More
How have complex brains evolved from simple circuits? Here we investigated brain region evolution at cell-type resolution in the cerebellar nuclei, the output structures of the cerebellum. Using single-nucleus RNA sequencing in mice, chickens, and humans, as well as STARmap spatial transcriptomic analysis and whole–central nervous system projection tracing, we identified a conserved cell-type set containing two region-specific excitatory neuron classes and three region-invariant inhibitory neuron classes. This set constitutes an archetypal cerebellar nucleus that was repeatedly duplicated to form new regions. The excitatory cell class that preferentially funnels information to lateral frontal cortices in mice becomes predominant in the massively expanded human lateral nucleus. Our data suggest a model of brain region evolution by duplication and divergence of entire cell-type sets. Less
Single-cell RNA-sequencing technologies are ideally placed to resolve intratumoral heterogeneity However the lack of coverage across key mutation hotspots has precluded the correlation of genetic and transcriptional readouts from the same single cell To overcome this we developed TARGET-seq a protocol for TARGETed high-sensitivity single-cell mutational analysis with extremely low allelic dropout rates parallel RNA SEQuencing and cell-surface proteomics Here we present a detailed step-by-step protocol for TARGET-seq including troubleshooting tips approaches for automation and methods for high-throughput multiplexing of libraries
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types Here we examine how specific transcriptomic types of mouse prefrontal cortex PFC projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks We found that most types projected to multiple targets and most targets received projections from multiple types except PFC PAG periaqueductal gray By comparing Ca activity of the molecularly homogeneous PFC PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice ... More
Single-cell transcriptomics has been widely applied to classify neurons in the mammalian brain, while systems neuroscience has historically analyzed the encoding properties of cortical neurons without considering cell types. Here we examine how specific transcriptomic types of mouse prefrontal cortex (PFC) projection neurons relate to axonal projections and encoding properties across multiple cognitive tasks. We found that most types projected to multiple targets, and most targets received projections from multiple types, except PFC→PAG (periaqueductal gray). By comparing Ca2+ activity of the molecularly homogeneous PFC→PAG type against two heterogeneous classes in several two-alternative choice tasks in freely moving mice, we found that all task-related signals assayed were qualitatively present in all examined classes. However, PAG-projecting neurons most potently encoded choice in cued tasks, whereas contralateral PFC-projecting neurons most potently encoded reward context in an uncued task. Thus, task signals are organized redundantly, but with clear quantitative biases across cells of specific molecular-anatomical characteristics. Less
The ongoing severe acute respiratory syndrome coronavirus SARS-CoV- pandemic has devastated the global economy and claimed more than million lives presenting an urgent global health crisis To identify host factors required for infection by SARS-CoV- and seasonal coronaviruses we designed a focused high-coverage CRISPR-Cas library targeting members of a recently published SARS-CoV- protein interactome We leveraged the compact nature of this library to systematically screen SARS-CoV- at two physiologically relevant temperatures along with three related coronaviruses human coronavirus E HCoV- E HCoV-NL and HCoV-OC allowing us to probe this interactome at a much higher resolution than genome-scale studies This approach ... More
The ongoing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has devastated the global economy and claimed more than 1.7 million lives, presenting an urgent global health crisis. To identify host factors required for infection by SARS-CoV-2 and seasonal coronaviruses, we designed a focused high-coverage CRISPR-Cas9 library targeting 332 members of a recently published SARS-CoV-2 protein interactome. We leveraged the compact nature of this library to systematically screen SARS-CoV-2 at two physiologically relevant temperatures along with three related coronaviruses (human coronavirus 229E [HCoV-229E], HCoV-NL63, and HCoV-OC43), allowing us to probe this interactome at a much higher resolution than genome-scale studies. This approach yielded several insights, including potential virus-specific differences in Rab GTPase requirements and glycosylphosphatidylinositol (GPI) anchor biosynthesis, as well as identification of multiple pan-coronavirus factors involved in cholesterol homeostasis. This coronavirus essentiality catalog could inform ongoing drug development efforts aimed at intercepting and treating coronavirus disease 2019 (COVID-19) and help prepare for future coronavirus outbreaks. 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
In this paper we applied EfficientNet a scalable deep convolution neural network with a custom data augmentation stage to a public protein crystallization image dataset called MARCO The MARCO dataset has protein crystallization images collected from several well-known institutions In our experiments EfficientNet outperformed the accuracies reported in the previous studies and it reached an overall testing and validation accuracy on the dataset Also EfficientNet achieved crystal detection accuracy in testing data which is significant improvement over existing studies
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
The role of gene expression during learning and in short-term memories has been studied extensively but less is known about remote memories which can persist for a lifetime Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning Out of a vast plasticity-coding space we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory Unexpectedly astrocytes and ... More
The role of gene expression during learning and in short-term memories has been studied extensively1,2,3, but less is known about remote memories, which can persist for a lifetime4. Here we used long-term contextual fear memory as a paradigm to probe the single-cell gene expression landscape that underlies remote memory storage in the medial prefrontal cortex. We found persistent activity-specific transcriptional alterations in diverse populations of neurons that lasted for weeks after fear learning. Out of a vast plasticity-coding space, we identified genes associated with membrane fusion that could have important roles in the maintenance of remote memory. Unexpectedly, astrocytes and microglia also acquired persistent gene expression signatures that were associated with remote memory, suggesting that they actively contribute to memory circuits. The discovery of gene expression programmes associated with remote memory engrams adds an important dimension of activity-dependent cellular states to existing brain taxonomy atlases and sheds light on the elusive mechanisms of remote memory storage. Less
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase LCP However almost all previous crystallization trials have used monoacylglycerols with - cis- -octadecanoyl -rac-glycerol MO being the most widely used lipid We now report that EROCOC mixed with w w cholesterol Fig serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A A receptor A AR The structures of EROCOC -matrix grown A AR crystals were determined at resolution by serial synchrotron rotation crystallography at a cryogenic temperature and at ... More
In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to − 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method. Less
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
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein and substrate engineering based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F A MD simulation further ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein and substrate engineering based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation could be achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternate binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that water entrance to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure-function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
CYP C from Nocardia farcinica is a P monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the -position Using protein engineering and substrate modifications based on the crystal structure of CYP C an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved Thus conversion of progesterone by mutant CYP C F A resulted in formation of the corresponding -hydroxylated product -deoxycorticosterone in addition to -hydroxylation Using MD simulation this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F A MD simulation ... More
CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation. Less
- - 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
PDGF VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine paracrine and endocrine mechanisms We investigated organ-specific metabolic roles of Drosophila PDGF VEGF-like factors Pvfs We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf signals to the Drosophila hepatocyte-like cells oenocytes to suppress lipid synthesis by activating the Pi K Akt TOR signaling cascade in the oenocytes Functionally this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis We find ... More
PDGF/VEGF ligands regulate a plethora of biological processes in multicellular organisms via autocrine, paracrine, and endocrine mechanisms. We investigated organ-specific metabolic roles of Drosophila PDGF/VEGF-like factors (Pvfs). We combine genetic approaches and single-nuclei sequencing to demonstrate that muscle-derived Pvf1 signals to the Drosophila hepatocyte-like cells/oenocytes to suppress lipid synthesis by activating the Pi3K/Akt1/TOR signaling cascade in the oenocytes. Functionally, this signaling axis regulates expansion of adipose tissue lipid stores in newly eclosed flies. Flies emerge after pupation with limited adipose tissue lipid stores and lipid level is progressively accumulated via lipid synthesis. We find that adult muscle-specific expression of pvf1 increases rapidly during this stage and that muscle-to-oenocyte Pvf1 signaling inhibits expansion of adipose tissue lipid stores as the process reaches completion. Our findings provide the first evidence in a metazoan of a PDGF/VEGF ligand acting as a myokine that regulates systemic lipid homeostasis by activating TOR in hepatocyte-like cells. Less
There is an increasing demand for rapid effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources Here we demonstrate a compact multimodal multiphoton microscope driven by a fiber-based ultrafast laser enabling excitation wavelengths at nm and nm for nonlinear optical imaging which simultaneously records second-harmonic generation third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography which we hope ... More
There is an increasing demand for rapid, effective methods to identify and detect protein micro- and nano-crystal suspensions for serial diffraction data collection at X-ray free-electron lasers or high-intensity micro-focus synchrotron radiation sources. Here, we demonstrate a compact multimodal, multiphoton microscope, driven by a fiber-based ultrafast laser, enabling excitation wavelengths at 775 nm and 1300 nm for nonlinear optical imaging, which simultaneously records second-harmonic generation, third-harmonic generation and three-photon excited ultraviolet fluorescence to identify and detect protein crystals with high sensitivity. The instrument serves as a valuable and important tool supporting sample scoring and sample optimization in biomolecular crystallography, which we hope will increase the capabilities and productivity of serial diffraction data collection in the future. Less
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia catecholaminergic polymorphic ventricular tachycardia CPVT In vivo calsequestrin multimerizes into filaments but a compelling atomic-resolution structure of a calsequestrin filament is lacking We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay We also report and characterize a novel disease-associated calsequestrin mutation S I which localizes to the filament-forming interface In addition we show that a previously reported dominant disease mutation K R maps to the same multimerization surface Both mutations disrupt filamentation suggesting that dominant disease ... More
Mutations in the calcium-binding protein calsequestrin cause a highly lethal familial arrhythmia, catecholaminergic polymorphic ventricular tachycardia (CPVT). In vivo, calsequestrin multimerizes into filaments, but a compelling atomic-resolution structure of a calsequestrin filament is lacking. We report a crystal structure of a cardiac calsequestrin filament with supporting mutation analysis provided by an in vitro fomentation assay. We also report and characterize a novel disease-associated calsequestrin mutation, S173I, which localizes to the filament-forming interface. In addition, we show that a previously reported dominant disease mutation, K180R, maps to the same multimerization surface. Both mutations disrupt filamentation, suggesting that dominant disease arises from defects in multimer formation. A ytterbium-derivatized structure pinpoints multiple credible calcium sites at filament-forming interfaces, explaining the atomic basis of calsequestrin filamentation in the presence of calcium. This work advances our understanding of calsequestrin biochemistry and provides a unifying structure-function molecular mechanism by which dominant-acting calsequestrin mutations provoke lethal arrhythmias. Less
Bright yellow BY- tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production This offers a range of benefits including the separation of the cell growth and heterologous gene expression lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding However low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants A number of molecular cell culture and down processing approaches have been made to overcome this problem Media development for the specific nutritional ... More
Bright yellow (BY-2) tobacco cells combined with the XVE chemically inducible system are one of the most promising plant-based platforms for recombinant protein production. This offers a range of benefits, including the separation of the cell growth and heterologous gene expression, lack of risk of infecting the end product with prions and human viruses or appropriate protein glycosylation and folding. However, low protein productivity remains a major obstacle that limits the extensive commercialization of bioproduction in plants. A number of molecular, cell culture and down processing approaches have been made to overcome this problem. Media development for the specific nutritional and hormonal requirements of transgenic plant cells is one of the most efficient cell-culture approaches. We optimized the induction medium towards recombinant protein production in BY-2 and demonstrated the usefulness of evolutionary medium optimization for high-yield protein production in liquid plant cultures. A reliable XVE/GFP model, parallel conducting experiments in a microscale on 96-well plates, and dedicated Gene Game evolutionary optimization software allowed for an effective search of 7611 possible solutions of 11-component media. Within the 4608 formulations tested, the Induct X medium was found with a significant 107.14% increase in protein expression in relation to the standard BY-2 medium. Less
Fungal aryl-alcohol oxidases AAOx are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids with concomitant production of hydrogen peroxide H O as by-product The H O can be provided to lignin degradation pathway a biotechnological property explored in biofuel production In the thermophilic fungus Thermothelomyces thermophilus formerly Myceliophthora thermophila just one AAOx was identified in the exo-proteome
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates one from the main group M and the other from the minor group O of HIV- we identify in the C-terminal domain CTD of integrase a new functional motif constituted by four non-contiguous amino acids N K N K Mutating the lysines abolishes integration through decreased -processing and inefficient nuclear import of reverse transcribed genomes Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration The number of charges in the motif appears ... More
Using coevolution-network interference based on the comparison of two phylogenetically distantly related isolates, one from the main group M and the other from the minor group O of HIV-1, we identify, in the C-terminal domain (CTD) of integrase, a new functional motif constituted by four non-contiguous amino acids (N222K240N254K273). Mutating the lysines abolishes integration through decreased 3’-processing and inefficient nuclear import of reverse transcribed genomes. Solution of the crystal structures of wt and mutated CTDs shows that the motif generates a positive surface potential that is important for integration. The number of charges in the motif appears more crucial than their position within the motif. Indeed, the positions of the K could be permutated or additional K could be inserted in the motif, generally without affecting integration per se. Despite this potential genetic flexibility, the NKNK arrangement is strictly conserved in natural sequences, indicative of an effective purifying selection exerted at steps other than integration. Accordingly, reverse transcription was reduced even in the mutants that retained wt integration levels, indicating that specifically the wt sequence is optimal for carrying out the multiple functions integrase exerts. We propose that the existence of several amino acids arrangements within the motif, with comparable efficiencies of integration per se, might have constituted an asset for the acquisition of additional functions during viral evolution. Less
Immuno-oncology approaches that utilize T cell receptors TCRs are becoming highly attractive because of their potential to target virtually all cellular proteins including cancer-specific epitopes via the recognition of peptide-human leukocyte antigen pHLA complexes presented at the cell surface However because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities efforts have been made to enhance their binding strength in some cases by several million-fold In this study we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors Additionally we performed molecular dynamics simulations ... More
Immuno-oncology approaches that utilize T cell receptors (TCRs) are becoming highly attractive because of their potential to target virtually all cellular proteins, including cancer-specific epitopes, via the recognition of peptide-human leukocyte antigen (pHLA) complexes presented at the cell surface. However, because natural TCRs generally recognize cancer-derived pHLAs with very weak affinities, efforts have been made to enhance their binding strength, in some cases by several million-fold. In this study, we investigated the mechanisms underpinning human TCR affinity enhancement by comparing the crystal structures of engineered enhanced affinity TCRs with those of their wild-type progenitors. Additionally, we performed molecular dynamics simulations to better understand the energetic mechanisms driving the affinity enhancements. These data demonstrate that supra-physiological binding affinities can be achieved without altering native TCR-pHLA binding modes via relatively subtle modifications to the interface contacts, often driven through the addition of buried hydrophobic residues. Individual energetic components of the TCR-pHLA interaction governing affinity enhancements were distinct and highly variable for each TCR, often resulting from additive, or knock-on, effects beyond the mutated residues. This comprehensive analysis of affinity-enhanced TCRs has important implications for the future rational design of engineered TCRs as efficacious and safe drugs for cancer treatment. Less
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases EHs reveals its resourceful microscopic machinery The whole-genome sequence of Streptomyces sp CBMAI an endophytic actinobacterium isolated from Citrus sinensis branches was explored by genome mining and a putative -epoxide hydrolase named B EPH and encoded by amino acids was selected for functional and structural studies The crystal structure of B EPH was obtained at a resolution of and ... More
The genus Streptomyces is characterized by the production of a wide variety of secondary metabolites with remarkable biological activities and broad antibiotic capabilities. The presence of an unprecedented number of genes encoding hydrolytic enzymes with industrial appeal such as epoxide hydrolases (EHs) reveals its resourceful microscopic machinery. The whole-genome sequence of Streptomyces sp. CBMAI 2042, an endophytic actinobacterium isolated from Citrus sinensis branches, was explored by genome mining, and a putative α/β-epoxide hydrolase named B1EPH2 and encoded by 344 amino acids was selected for functional and structural studies. The crystal structure of B1EPH2 was obtained at a resolution of 2.2 Å and it was found to have a similar fold to other EHs, despite its hexameric quaternary structure, which contrasts with previously solved dimeric and monomeric EH structures. While B1EPH2 has a high sequence similarity to EHB from Mycobacterium tuberculosis, its cavity is similar to that of human EH. A group of 12 aromatic and aliphatic racemic epoxides were assayed to determine the activity of B1EPH2; remarkably, this enzyme was able to hydrolyse all the epoxides to the respective 1,2-diols, indicating a wide-range substrate scope acceptance. Moreover, the (R)- and (S)-enantiomers of styrene oxide, epichlorohydrin and 1,2-epoxybutane were used to monitor enantiopreference. Taken together, the functional and structural analyses indicate that this enzyme is an attractive biocatalyst for future biotechnological applications. Less
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
Ageing is characterised by cellular senescence leading to imbalanced tissue maintenance cell death and compromised organ function This is first observed in the thymus the primary lymphoid organ that generates and selects T cells However the molecular and cellular mechanisms underpinning these ageing processes remain unclear Here we show that mouse ageing leads to less efficient T cell selection decreased self-antigen representation and increased T cell receptor repertoire diversity Using a combination of single-cell RNA-seq and lineage-tracing we find that progenitor cells are the principal targets of ageing whereas the function of individual mature thymic epithelial cells is compromised only ... More
Ageing is characterised by cellular senescence, leading to imbalanced tissue maintenance, cell death and compromised organ function. This is first observed in the thymus, the primary lymphoid organ that generates and selects T cells. However, the molecular and cellular mechanisms underpinning these ageing processes remain unclear. Here, we show that mouse ageing leads to less efficient T cell selection, decreased self-antigen representation and increased T cell receptor repertoire diversity. Using a combination of single-cell RNA-seq and lineage-tracing, we find that progenitor cells are the principal targets of ageing, whereas the function of individual mature thymic epithelial cells is compromised only modestly. Specifically, an early-life precursor cell population, retained in the mouse cortex postnatally, is virtually extinguished at puberty. Concomitantly, a medullary precursor cell quiesces, thereby impairing maintenance of the medullary epithelium. Thus, ageing disrupts thymic progenitor differentiation and impairs the core immunological functions of the thymus. Less
Lung cancer the leading cause of cancer mortality exhibits heterogeneity that enables adaptability limits therapeutic success and remains incompletely understood Single-cell RNA sequencing scRNA-seq of metastatic lung cancer was performed using clinical biopsies obtained from patients before and during targeted therapy Over cancer and tumor microenvironment TME single-cell profiles exposed a rich and dynamic tumor ecosystem scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically Cancer cells surviving therapy as residual disease RD expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition whereas those present at on-therapy progressive disease PD upregulated kynurenine plasminogen and gap-junction pathways ... More
Lung cancer, the leading cause of cancer mortality, exhibits heterogeneity that enables adaptability, limits therapeutic success, and remains incompletely understood. Single-cell RNA sequencing (scRNA-seq) of metastatic lung cancer was performed using 49 clinical biopsies obtained from 30 patients before and during targeted therapy. Over 20,000 cancer and tumor microenvironment (TME) single-cell profiles exposed a rich and dynamic tumor ecosystem. scRNA-seq of cancer cells illuminated targetable oncogenes beyond those detected clinically. Cancer cells surviving therapy as residual disease (RD) expressed an alveolar-regenerative cell signature suggesting a therapy-induced primitive cell-state transition, whereas those present at on-therapy progressive disease (PD) upregulated kynurenine, plasminogen, and gap-junction pathways. Active T-lymphocytes and decreased macrophages were present at RD and immunosuppressive cell states characterized PD. Biological features revealed by scRNA-seq were biomarkers of clinical outcomes in independent cohorts. This study highlights how therapy-induced adaptation of the multi-cellular ecosystem of metastatic cancer shapes clinical outcomes. 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
In biology it is often critical to determine the identity of an organism and phenotypic traits of interest Whole-genome sequencing can be useful for this but has limited power for trait prediction However we can take advantage of the inherent information content of phenotypes to bypass these limitations We demonstrate in clinical and environmental bacterial isolates that growth dynamics in standardized conditions can differentiate between genotypes even among strains from the same species We find that for pairs of isolates there is little correlation between genetic distance according to phylogenetic analysis and phenotypic distance as determined by growth dynamics This ... More
In biology, it is often critical to determine the identity of an organism and phenotypic traits of interest. Whole-genome sequencing can be useful for this but has limited power for trait prediction. However, we can take advantage of the inherent information content of phenotypes to bypass these limitations. We demonstrate, in clinical and environmental bacterial isolates, that growth dynamics in standardized conditions can differentiate between genotypes, even among strains from the same species. We find that for pairs of isolates, there is little correlation between genetic distance, according to phylogenetic analysis, and phenotypic distance, as determined by growth dynamics. This absence of correlation underscores the challenge in using genomics to infer phenotypes and vice versa. Bypassing this complexity, we show that growth dynamics alone can robustly predict antibiotic responses. These findings are a foundation for a method to identify traits not easily traced to a genetic mechanism. Less
T cell-mediated immunity is governed primarily by T cell receptor TCR recognition of peptide-human leukocyte antigen pHLA complexes and is essential for immunosurveillance and disease control This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region CDR loops and whereas peptide selectivity is guided by direct interactions with the TCR CDR loops Here we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A MAGE-A The TCR bound pHLA in a position shifted toward the peptide's N terminus This enabled the ... More
T cell-mediated immunity is governed primarily by T cell receptor (TCR) recognition of peptide-human leukocyte antigen (pHLA) complexes and is essential for immunosurveillance and disease control. This interaction is generally stabilized by interactions between the HLA surface and TCR germline-encoded complementarity-determining region (CDR) loops 1 and 2, whereas peptide selectivity is guided by direct interactions with the TCR CDR3 loops. Here, we solved the structure of a newly identified TCR in complex with a clinically relevant peptide derived from the cancer testis antigen melanoma antigen-A4 (MAGE-A4). The TCR bound pHLA in a position shifted toward the peptide's N terminus. This enabled the TCR to achieve peptide selectivity via an indirect mechanism, whereby the TCR sensed the first residue of the peptide through HLA residue Trp-167, which acted as a tunable gateway. Amino acid substitutions at peptide position 1 predicted to alter the HLA Trp-167 side-chain conformation abrogated TCR binding, indicating that this indirect binding mechanism is essential for peptide recognition. These findings extend our understanding of the molecular rules that underpin antigen recognition by TCRs and have important implications for the development of TCR-based therapies. Less
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins However to date there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries To resolve this limitation we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications ... More
Baculovirus mediated-insect cell expression systems have been widely used for producing heterogeneous proteins. However, to date, there is still the lack of an easy-to-manipulate system that enables the high-throughput protein characterization in insect cells by taking advantage of large existing Gateway clone libraries. To resolve this limitation, we have constructed a suite of Gateway-compatible pIEx-derived baculovirus expression vectors that allow the rapid and cost-effective construction of expression clones for mass parallel protein expression in insect cells. This vector collection also supports the attachment of a variety of fusion tags to target proteins to meet the needs for different research applications. We first demonstrated the utility of these vectors for protein expression and purification using a set of 40 target proteins of various sizes, cellular localizations and host organisms. We then established a scalable pipeline coupled with the SONICC and TEM techniques to screen for microcrystal formation within living insect cells. Using this pipeline, we successfully identified microcrystals for ~ 16% of the tested protein set, which can be potentially used for structure elucidation by X-ray crystallography. In summary, we have established a versatile pipeline enabling parallel gene cloning, protein expression and purification, and in vivo microcrystal screening for structural studies. Less