Molecular mapping of the RNA Cap 2'-O-methyltransferase activation interface between severe acute respiratory syndrome coronavirus nsp10 and nsp16.
Several protein-protein interactions within the SARS-CoV proteome have been identified, one of them being between non-structural proteins nsp10 and nsp16. In this work, we have mapped key residues on the nsp10 surface involved in this interaction. Alanine-scanning mutagenesis, bioinformatics, and molecular modeling were used to identify several "hot spots," such as Val(42), Met(44), Ala(71), Lys(93), Gly(94), and Tyr(96), forming a continuous protein-protein surface of about 830 Å(2), bearing very conserved amino acids among coronaviruses. Because nsp16 carries RNA cap 2'-O-methyltransferase (2'O-MTase) activity only in the presence of its interacting partner nsp10 (Bouvet, M., Debarnot, C., Imbert, I., Selisko, B., Snijder, E. J., Canard, B., and Decroly, E. (2010) PLoS Pathog. 6, e1000863), functional consequences of mutations on this surface were evaluated biochemically. Most changes that disrupted the nsp10-nsp16 interaction without structural perturbations were shown to abrogate stimulation of nsp16 RNA cap 2'O-MTase activity. More strikingly, the Y96A mutation abrogates stimulation of nsp16 2'O-MTase activity, whereas Y96F overstimulates it. Thus, the nsp10-nsp16 interface may represent an attractive target for antivirals against human and animal pathogenic coronaviruses.
Lugari
Adrien
A
From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Betzi
Stephane
S
From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Decroly
Etienne
E
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Bonnaud
Emmanuel
E
INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
Hermant
Aurélie
A
INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
Guillemot
Jean-Claude
JC
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Debarnot
Claire
C
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Borg
Jean-Paul
JP
INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France.
Bouvet
Mickaël
M
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Canard
Bruno
B
Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d'Aix-Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France.
Morelli
Xavier
X
From the CNRS and Aix-Marseille Universités, IMR Laboratory (UPR 3243), Institut de Microbiologie de la Méditérannée, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: morelli@ifr88.cnrs-mrs.fr.
Lécine
Patrick
P
INSERM, UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille F-13009, France; Institut Paoli-Calmettes, Marseille F-13009, France; Université Méditerranée, Marseille F-13007, France. Electronic address: patrick.lecine@inserm.fr.
D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D008780:Methyltransferases / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis; D020125:Mutation, Missense; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C032117:cap I RNA (nucleoside-2'-)methyltransferase