Giuseppe
Emeritus
[Source: PLoS Pathogens, full text: (LINK).]
Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex
Etienne Decroly<SUP>1</SUP><SUP>*</SUP>, Claire Debarnot<SUP>1</SUP>, Fran?ois Ferron<SUP>1</SUP>, Mickael Bouvet<SUP>1</SUP>, Bruno Coutard<SUP>1</SUP>, Isabelle Imbert<SUP>1</SUP>, Laure Gluais<SUP>1</SUP>, Nicolas Papageorgiou<SUP>1</SUP>, Andrew Sharff<SUP>2</SUP>, G?rard Bricogne<SUP>2</SUP>, Miguel Ortiz-Lombardia<SUP>1</SUP>, Julien Lescar<SUP>1</SUP><SUP>,</SUP><SUP>3</SUP>, Bruno Canard<SUP>1</SUP><SUP>*</SUP>
1 Centre National de la Recherche Scientifique and Universit? de la M?diterran?e, UMR 6098, Architecture et Fonction des Macromol?cules Biologiques, Marseille, France, 2 Global Phasing Ltd., Sheraton House, Castle Park, Cambridge, United Kingdom, 3 School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore
Abstract
Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 ? resolution, which shows nsp10 bound to nsp16 through a ~930 ?<SUP>2</SUP> surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Author Summary
A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ~930 ?<SUP>2</SUP> activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Citation: Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, et al. (2011) Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex. PLoS Pathog 7(5): e1002059. doi:10.1371/journal.ppat.1002059
Editor: F?lix A. Rey, Institut Pasteur, France
Received: December 7, 2010; Accepted: March 23, 2011; Published: May 26, 2011
Copyright: ? 2011 Decroly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in its initial phase, by the Euro-Asian SARS-DTV Network (SP22-CT-2004-511064) from the European Commission specific research and technological development Programme ?Integrating and strengthening the European Research area.? It was subsequently supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework and the French National Research agency, under reference ?ANR-08-MIEN-032,? and subsequently the Fondation pour la Recherche M?dicale (Programme ?quipe FRM), and the Direction G?n?rale de l'Armement (contrat n?07co404). MB has a fellowship from the Direction G?n?rale de l'Armement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: etienne.decroly@afmb.univ-mrs.fr (ED); bruno.canard@afmb.univ-mrs.fr (BC)
- -------Etienne Decroly<SUP>1</SUP><SUP>*</SUP>, Claire Debarnot<SUP>1</SUP>, Fran?ois Ferron<SUP>1</SUP>, Mickael Bouvet<SUP>1</SUP>, Bruno Coutard<SUP>1</SUP>, Isabelle Imbert<SUP>1</SUP>, Laure Gluais<SUP>1</SUP>, Nicolas Papageorgiou<SUP>1</SUP>, Andrew Sharff<SUP>2</SUP>, G?rard Bricogne<SUP>2</SUP>, Miguel Ortiz-Lombardia<SUP>1</SUP>, Julien Lescar<SUP>1</SUP><SUP>,</SUP><SUP>3</SUP>, Bruno Canard<SUP>1</SUP><SUP>*</SUP>
1 Centre National de la Recherche Scientifique and Universit? de la M?diterran?e, UMR 6098, Architecture et Fonction des Macromol?cules Biologiques, Marseille, France, 2 Global Phasing Ltd., Sheraton House, Castle Park, Cambridge, United Kingdom, 3 School of Biological Sciences, Nanyang Technological University, Singapore, Republic of Singapore
Abstract
Cellular and viral S-adenosylmethionine-dependent methyltransferases are involved in many regulated processes such as metabolism, detoxification, signal transduction, chromatin remodeling, nucleic acid processing, and mRNA capping. The Severe Acute Respiratory Syndrome coronavirus nsp16 protein is a S-adenosylmethionine-dependent (nucleoside-2′-O)-methyltransferase only active in the presence of its activating partner nsp10. We report the nsp10/nsp16 complex structure at 2.0 ? resolution, which shows nsp10 bound to nsp16 through a ~930 ?<SUP>2</SUP> surface area in nsp10. Functional assays identify key residues involved in nsp10/nsp16 association, and in RNA binding or catalysis, the latter likely through a SN2-like mechanism. We present two other crystal structures, the inhibitor Sinefungin bound in the S-adenosylmethionine binding pocket and the tighter complex nsp10(Y96F)/nsp16, providing the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Author Summary
A novel coronavirus emerged in 2003 and was identified as the etiological agent of the deadly disease called Severe Acute Respiratory Syndrome. This coronavirus replicates and transcribes its giant genome using sixteen non-structural proteins (nsp1-16). Viral RNAs are capped to ensure stability, efficient translation, and evading the innate immunity system of the host cell. The nsp16 protein is a RNA cap modifying enzyme only active in the presence of its activating partner nsp10. We have crystallized the nsp10/16 complex and report its crystal structure at atomic resolution. Nsp10 binds to nsp16 through a ~930 ?<SUP>2</SUP> activation surface area in nsp10, and the resulting complex exhibits RNA cap (nucleoside-2′-O)-methyltransferase activity. We have performed mutational and functional assays to identify key residues involved in catalysis and/or in RNA binding, and in the association of nsp10 to nsp16. We present two additional crystal structures, that of the known inhibitor Sinefungin bound in the SAM binding pocket, and that of a tighter complex made of the mutant nsp10(Y96F) bound to nsp16. Our study provides a basis for antiviral drug design as well as the first structural insight into the regulation of RNA capping enzymes in (+)RNA viruses.
Citation: Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, et al. (2011) Crystal Structure and Functional Analysis of the SARS-Coronavirus RNA Cap 2′-O-Methyltransferase nsp10/nsp16 Complex. PLoS Pathog 7(5): e1002059. doi:10.1371/journal.ppat.1002059
Editor: F?lix A. Rey, Institut Pasteur, France
Received: December 7, 2010; Accepted: March 23, 2011; Published: May 26, 2011
Copyright: ? 2011 Decroly et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported, in its initial phase, by the Euro-Asian SARS-DTV Network (SP22-CT-2004-511064) from the European Commission specific research and technological development Programme ?Integrating and strengthening the European Research area.? It was subsequently supported by the VIZIER integrated project (LSHG-CT-2004-511960) of the European Union 6th Framework and the French National Research agency, under reference ?ANR-08-MIEN-032,? and subsequently the Fondation pour la Recherche M?dicale (Programme ?quipe FRM), and the Direction G?n?rale de l'Armement (contrat n?07co404). MB has a fellowship from the Direction G?n?rale de l'Armement. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: etienne.decroly@afmb.univ-mrs.fr (ED); bruno.canard@afmb.univ-mrs.fr (BC)