Hepatitis B virus core protein phosphorylation: Identification of the SRPK1 target sites and impact of their occupancy on RNA binding and capsid structure

International audienceHepatitis B virus (HBV) replicates its 3 kb DNA genome through capsid-internal reverse transcription, initiated by assembly of 120 core protein (HBc) dimers around a complex of viral pregenomic (pg) RNA and polymerase. Following synthesis of relaxed circular (RC) DNA capsids can be enveloped and secreted as stable virions. Upon infection of a new cell, however, the capsid disintegrates to release the RC-DNA into the nucleus for conversion into covalently closed circular (ccc) DNA. HBc´s interactions with nucleic acids are mediated by an arginine-rich C terminal domain (CTD) with intrinsically strong non-specific RNA binding activity. Adaptation to the changing demands for nucleic acid binding during the viral life cycle is thought to involve dynamic phosphorylation / dephosphorylation events. However, neither the relevant enzymes nor their target sites in HBc are firmly established. Here we developed a bacterial coexpression system enabling access to definably phosphorylated HBc. Combining Phos-tag gel electrophoresis, mass spectrometry and mutagenesis we identified seven of the eight hydroxy amino acids in the CTD as target sites for serine-argi-nine rich protein kinase 1 (SRPK1); fewer sites were phosphorylated by PKA and PKC. Phosphorylation of all seven sites reduced nonspecific RNA encapsidation as drastically as deletion of the entire CTD and altered CTD surface accessibility, without major structure changes in the capsid shell. The bulk of capsids from human hepatoma cells was similarly highly, yet non-identically, phosphorylated as by SRPK1. While not proving SRPK1 as the infection-relevant HBc kinase the data suggest a mechanism whereby high-level HBc phos-phorylation principally suppresses RNA binding whereas one or few strategic dephosphory-lation events enable selective packaging of the pgRNA/polymerase complex. The tools developed in this study should greatly facilitate the further deciphering of the role of HBc phosphorylation in HBV infection and its evaluation as a potential new therapeutic target. PLOS Pathogens | https://doi.org/10.1371/journal.ppat

Combining Cell-Free Protein Synthesis and NMR Into a Tool to Study Capsid Assembly Modulation

International audienceModulation of capsid assembly by small molecules has become a central concept in the fight against viral infection. Proper capsid assembly is crucial to form the high molecular weight structures that protect the viral genome and that, often in concert with the envelope, allow for cell entry and fusion. Atomic details underlying assembly modulation are generally studied using preassembled protein complexes, while the activity of assembly modulators during assembly remains largely open and poorly understood, as necessary tools are lacking. We here use the full-length hepatitis B virus (HBV) capsid protein (Cp183) as a model to present a combination of cell-free protein synthesis and solid-state NMR as an approach which shall open the possibility to produce and analyze the formation of higher-order complexes directly on exit from the ribosome. We demonstrate that assembled capsids can be synthesized in amounts sufficient for structural studies, and show that addition of assembly modulators to the cell-free reaction produces objects similar to those obtained by addition of the compounds to preformed Cp183 capsids. These results establish the cell-free system as a tool for the study of capsid assembly modulation directly after synthesis by the ribosome, and they open the perspective of assessing the impact of natural or synthetic compounds, or even enzymes that perform post-translational modifications, on capsids structures

Comprehensive Fragment Screening of the SARS-CoV-2 Proteome Explores Novel Chemical Space for Drug Development

12 pags., 4 figs., 3 tabs.SARS-CoV-2 (SCoV2) and its variants of concern pose serious challenges to the public health. The variants increased challenges to vaccines, thus necessitating for development of new intervention strategies including anti-virals. Within the international Covid19-NMR consortium, we have identified binders targeting the RNA genome of SCoV2. We established protocols for the production and NMR characterization of more than 80 % of all SCoV2 proteins. Here, we performed an NMR screening using a fragment library for binding to 25 SCoV2 proteins and identified hits also against previously unexplored SCoV2 proteins. Computational mapping was used to predict binding sites and identify functional moieties (chemotypes) of the ligands occupying these pockets. Striking consensus was observed between NMR-detected binding sites of the main protease and the computational procedure. Our investigation provides novel structural and chemical space for structure-based drug design against the SCoV2 proteome.Work at BMRZ is supported by the state of Hesse. Work in Covid19-NMR was supported by the Goethe Corona Funds, by the IWBEFRE-program 20007375 of state of Hesse, the DFG through CRC902: “Molecular Principles of RNA-based regulation.” and through infrastructure funds (project numbers: 277478796, 277479031, 392682309, 452632086, 70653611) and by European Union’s Horizon 2020 research and innovation program iNEXT-discovery under grant agreement No 871037. BY-COVID receives funding from the European Union’s Horizon Europe Research and Innovation Programme under grant agreement number 101046203. “INSPIRED” (MIS 5002550) project, implemented under the Action “Reinforcement of the Research and Innovation Infrastructure,” funded by the Operational Program “Competitiveness, Entrepreneurship and Innovation” (NSRF 2014–2020) and co-financed by Greece and the EU (European Regional Development Fund) and the FP7 REGPOT CT-2011-285950—“SEE-DRUG” project (purchase of UPAT’s 700 MHz NMR equipment). The support of the CERM/CIRMMP center of Instruct-ERIC is gratefully acknowledged. This work has been funded in part by a grant of the Italian Ministry of University and Research (FISR2020IP_02112, ID-COVID) and by Fondazione CR Firenze. A.S. is supported by the Deutsche Forschungsgemeinschaft [SFB902/B16, SCHL2062/2-1] and the Johanna Quandt Young Academy at Goethe [2019/AS01]. M.H. and C.F. thank SFB902 and the Stiftung Polytechnische Gesellschaft for the Scholarship. L.L. work was supported by the French National Research Agency (ANR, NMR-SCoV2-ORF8), the Fondation de la Recherche Médicale (FRM, NMR-SCoV2-ORF8), FINOVI and the IR-RMN-THC Fr3050 CNRS. Work at UConn Health was supported by grants from the US National Institutes of Health (R01 GM135592 to B.H., P41 GM111135 and R01 GM123249 to J.C.H.) and the US National Science Foundation (DBI 2030601 to J.C.H.). Latvian Council of Science Grant No. VPP-COVID-2020/1-0014. National Science Foundation EAGER MCB-2031269. This work was supported by the grant Krebsliga KFS-4903-08-2019 and SNF-311030_192646 to J.O. P.G. (ITMP) The EOSC Future project is co-funded by the European Union Horizon Programme call INFRAEOSC-03-2020—Grant Agreement Number 101017536. Open Access funding enabled and organized by Projekt DEALPeer reviewe

Large-Scale Recombinant Production of the SARS-CoV-2 Proteome for High-Throughput and Structural Biology Applications

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form

NMR study of bacterial L,D-transpeptidases : structure, dynamics and insights into their inhibition by beta-lacams

L'étape finale de biosynthèse du peptidoglycane est catalysée par les D,D-transpeptidases (PBPs), l'une des cibles principales des antibiotiques de type beta-lactame. Récemment, il a été montré qu'une nouvelle classe d'enzymes, les L,D-transpeptidases (LDts), permet de contourner l'inhibition des PBPs. Ces LDts ont été identifiées tant dans des bactéries résistantes aux beta-lactames que dans des formes dormantes de Mycobacterium tuberculosis. Les seuls beta-lactames capables de les inhiber, les carbapénèmes, forment une liaison covalente avec la cystéine catalytique des LDts. Ni le mécanisme de cette inactivation, ni la spécificité de ces enzymes pour les carbapénèmes ne sont toutefois expliqués à ce jour. Le but du présent travail consiste en l'investigation par RMN du mécanisme d'acylation des LDts par ces antibiotiques. Dans ce contexte, la première partie de cette thèse s'intéresse à la compréhension actuelle de l'émergence de ce phénomène de résistance. La seconde partie traite des principes de la RMN et des implémentations développées pour étudier la structure, la thermodynamique et la dynamique des LDts. La troisième et dernière partie démontre le succès de l'approche RMN dans l'étude des diverses étapes de la réaction d'acylation, à travers une étude détaillée de l'apoenzyme, de complexes non covalents avec différents beta-lactames, et de l'enzyme acylée par un carbapénème. Au cours de cette étude, la structure du site actif de l'apoenzyme de Bacillus subtilis a été affinée par rapport à une étude cristallographique antérieure. Pour cette enzyme et son pendant chez Enterococcus faecium, nous avons démontré que la spécificité pour les carbapénèmes n'intervient pas au stade de la formation du complexe non covalent. Pour finir, la formation de la liaison covalente entre LDt et carbapénème induit un réarrangement conformationnel substantiel et une augmentation de la flexibilité de l'enzyme.The final cross-linking step of the peptidoglycan synthesis is usually catalyzed by D,D-transpeptidases (PBPs), one of the main targets of beta-lactam antibiotics. Recently, it was shown that these PBPs can be by-passed by a novel class of enzymes, the L,D-transpeptidases (LDts), identified in beta-lactam-resistant bacteria as well as in dormant forms of Mycobacterium tuberculosis. The only beta-lactams enable to inactivate these enzymes belong to the carbapenem class. The beta-lactam ring of this antibiotic family then covalently binds the catalytic cysteine of the LDt. Neither the mechanism of this reaction nor the specificity for carbapenems are yet understood. The aim of the present work is to investigate the acylation mechanism of LDts with carbapenems by NMR. In this context, the first part of this thesis focuses on the current biological understanding of the emergence of this resistance pathway. The second part deals with the NMR principles and the implementations developed to study the structure, thermodynamics and dynamics of LDts. The third part demonstrates that NMR is successful in studying all the steps of the acylation reaction. For this purpose, the LDt apoenzyme, the non-covalent complex with various beta-lactams, and the LDt-carbapenem acylenzyme were thoroughly investigated. The structure of the active site of the Bacillus subtilis apoenzyme was refined with respect to a previous crystallographic study. For the latter and the Enterococcus faecium enzymes, we showed that the carbapenem specificity does not occur at the stage of the non-covalent binding. In contrast to non-covalent interactions, the formation of the covalent bond between LDts and carbapenems induces substantial conformational rearrangement and increased flexibility in the enzyme

Etudes par RMN des L,D-transpeptidases bactériennes : structure, dynamique et compréhension de leur inhibition par les beta-lactames

The final cross-linking step of the peptidoglycan synthesis is usually catalyzed by D,D-transpeptidases (PBPs), one of the main targets of beta-lactam antibiotics. Recently, it was shown that these PBPs can be by-passed by a novel class of enzymes, the L,D-transpeptidases (LDts), identified in beta-lactam-resistant bacteria as well as in dormant forms of Mycobacterium tuberculosis. The only beta-lactams enable to inactivate these enzymes belong to the carbapenem class. The beta-lactam ring of this antibiotic family then covalently binds the catalytic cysteine of the LDt. Neither the mechanism of this reaction nor the specificity for carbapenems are yet understood. The aim of the present work is to investigate the acylation mechanism of LDts with carbapenems by NMR. In this context, the first part of this thesis focuses on the current biological understanding of the emergence of this resistance pathway. The second part deals with the NMR principles and the implementations developed to study the structure, thermodynamics and dynamics of LDts. The third part demonstrates that NMR is successful in studying all the steps of the acylation reaction. For this purpose, the LDt apoenzyme, the non-covalent complex with various beta-lactams, and the LDt-carbapenem acylenzyme were thoroughly investigated. The structure of the active site of the Bacillus subtilis apoenzyme was refined with respect to a previous crystallographic study. For the latter and the Enterococcus faecium enzymes, we showed that the carbapenem specificity does not occur at the stage of the non-covalent binding. In contrast to non-covalent interactions, the formation of the covalent bond between LDts and carbapenems induces substantial conformational rearrangement and increased flexibility in the enzyme.L'étape finale de biosynthèse du peptidoglycane est catalysée par les D,D-transpeptidases (PBPs), l'une des cibles principales des antibiotiques de type beta-lactame. Récemment, il a été montré qu'une nouvelle classe d'enzymes, les L,D-transpeptidases (LDts), permet de contourner l'inhibition des PBPs. Ces LDts ont été identifiées tant dans des bactéries résistantes aux beta-lactames que dans des formes dormantes de Mycobacterium tuberculosis. Les seuls beta-lactames capables de les inhiber, les carbapénèmes, forment une liaison covalente avec la cystéine catalytique des LDts. Ni le mécanisme de cette inactivation, ni la spécificité de ces enzymes pour les carbapénèmes ne sont toutefois expliqués à ce jour. Le but du présent travail consiste en l'investigation par RMN du mécanisme d'acylation des LDts par ces antibiotiques. Dans ce contexte, la première partie de cette thèse s'intéresse à la compréhension actuelle de l'émergence de ce phénomène de résistance. La seconde partie traite des principes de la RMN et des implémentations développées pour étudier la structure, la thermodynamique et la dynamique des LDts. La troisième et dernière partie démontre le succès de l'approche RMN dans l'étude des diverses étapes de la réaction d'acylation, à travers une étude détaillée de l'apoenzyme, de complexes non covalents avec différents beta-lactames, et de l'enzyme acylée par un carbapénème. Au cours de cette étude, la structure du site actif de l'apoenzyme de Bacillus subtilis a été affinée par rapport à une étude cristallographique antérieure. Pour cette enzyme et son pendant chez Enterococcus faecium, nous avons démontré que la spécificité pour les carbapénèmes n'intervient pas au stade de la formation du complexe non covalent. Pour finir, la formation de la liaison covalente entre LDt et carbapénème induit un réarrangement conformationnel substantiel et une augmentation de la flexibilité de l'enzyme

Etudes par RMN des L,D-transpeptidases bactériennes : structure, dynamique et compréhension de leur inhibition par les beta-lactames

The final cross-linking step of the peptidoglycan synthesis is usually catalyzed by D,D-transpeptidases (PBPs), one of the main targets of beta-lactam antibiotics. Recently, it was shown that these PBPs can be by-passed by a novel class of enzymes, the L,D-transpeptidases (LDts), identified in beta-lactam-resistant bacteria as well as in dormant forms of Mycobacterium tuberculosis. The only beta-lactams enable to inactivate these enzymes belong to the carbapenem class. The beta-lactam ring of this antibiotic family then covalently binds the catalytic cysteine of the LDt. Neither the mechanism of this reaction nor the specificity for carbapenems are yet understood. The aim of the present work is to investigate the acylation mechanism of LDts with carbapenems by NMR. In this context, the first part of this thesis focuses on the current biological understanding of the emergence of this resistance pathway. The second part deals with the NMR principles and the implementations developed to study the structure, thermodynamics and dynamics of LDts. The third part demonstrates that NMR is successful in studying all the steps of the acylation reaction. For this purpose, the LDt apoenzyme, the non-covalent complex with various beta-lactams, and the LDt-carbapenem acylenzyme were thoroughly investigated. The structure of the active site of the Bacillus subtilis apoenzyme was refined with respect to a previous crystallographic study. For the latter and the Enterococcus faecium enzymes, we showed that the carbapenem specificity does not occur at the stage of the non-covalent binding. In contrast to non-covalent interactions, the formation of the covalent bond between LDts and carbapenems induces substantial conformational rearrangement and increased flexibility in the enzyme.L'étape finale de biosynthèse du peptidoglycane est catalysée par les D,D-transpeptidases (PBPs), l'une des cibles principales des antibiotiques de type beta-lactame. Récemment, il a été montré qu'une nouvelle classe d'enzymes, les L,D-transpeptidases (LDts), permet de contourner l'inhibition des PBPs. Ces LDts ont été identifiées tant dans des bactéries résistantes aux beta-lactames que dans des formes dormantes de Mycobacterium tuberculosis. Les seuls beta-lactames capables de les inhiber, les carbapénèmes, forment une liaison covalente avec la cystéine catalytique des LDts. Ni le mécanisme de cette inactivation, ni la spécificité de ces enzymes pour les carbapénèmes ne sont toutefois expliqués à ce jour. Le but du présent travail consiste en l'investigation par RMN du mécanisme d'acylation des LDts par ces antibiotiques. Dans ce contexte, la première partie de cette thèse s'intéresse à la compréhension actuelle de l'émergence de ce phénomène de résistance. La seconde partie traite des principes de la RMN et des implémentations développées pour étudier la structure, la thermodynamique et la dynamique des LDts. La troisième et dernière partie démontre le succès de l'approche RMN dans l'étude des diverses étapes de la réaction d'acylation, à travers une étude détaillée de l'apoenzyme, de complexes non covalents avec différents beta-lactames, et de l'enzyme acylée par un carbapénème. Au cours de cette étude, la structure du site actif de l'apoenzyme de Bacillus subtilis a été affinée par rapport à une étude cristallographique antérieure. Pour cette enzyme et son pendant chez Enterococcus faecium, nous avons démontré que la spécificité pour les carbapénèmes n'intervient pas au stade de la formation du complexe non covalent. Pour finir, la formation de la liaison covalente entre LDt et carbapénème induit un réarrangement conformationnel substantiel et une augmentation de la flexibilité de l'enzyme

Solid-State NMR for Studying the Structure and Dynamics of Viral Assemblies

Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years

1H, 15N and 13C backbone and side chain solution NMR assignments of the truncated small hepatitis delta antigen Delta60-S-HDAg

International audienceHepatitis D virus (HDV) is a defective virus that relies on hepatitis B virus envelope proteins to complete its replication cycle. The HDV genome contains two isoforms of hepatitis delta antigen: the small and the large hepatitis delta antigens (S-and L-HDAg). Here we report the 1 H , 13 C and 15 N backbone and side chain resonance assignments of an N-terminally truncated form of S-HDAg (S D60), which lacks the 1-60 oligomerization domain. We derived secondary structures based on NMR chemical shifts, which will be used in further structural and functional studies. We show that S D60 is partially disordered, and that the central structured part contains two well-defined α-helices of 22 and 17 residues, respectively. A temperature titration allowed to identify the residues involved in hydrogen bonds

Solid-state NMR for studying the structure and dynamics of viral assemblies

Structural virology reveals the architecture underlying infection. While notably electron microscopy images have provided an atomic view on viruses which profoundly changed our understanding of these assemblies incapable of independent life, spectroscopic techniques like NMR enter the field with their strengths in detailed conformational analysis and investigation of dynamic behavior. Typically, the large assemblies represented by viral particles fall in the regime of biological high-resolution solid-state NMR, able to follow with high sensitivity the path of the viral proteins through their interactions and maturation steps during the viral life cycle. We here trace the way from first solid-state NMR investigations to the state-of-the-art approaches currently developing, including applications focused on HIV, HBV, HCV and influenza, and an outlook to the possibilities opening in the coming years