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Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, Imbert I, Gluais L, Papageorgiou N, Sharff A, Bricogne G, Ortiz-Lombardia M, Lescar J, Canard B - PLoS Pathog. (2011)

Bottom Line: We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų 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.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique and Université de la Méditerranée, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Marseille, France. etienne.decroly@afmb.univ-mrs.fr

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 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų 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.

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Related in: MedlinePlus

Position and coordination of a metal ion.Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.
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ppat-1002059-g004: Position and coordination of a metal ion.Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.

Mentions: We recently reported[16] that nsp10/nsp16 MTase activity requires Mg2+. Although the crystallization buffer contains Mg2+, we were unable to locate any such cation in the nsp16 active site. In enzyme activity assays, the Mg2+ ion can be substituted by Mn2+ or Ca2+, but not Zn2+ (data not shown, see also[16]). A peak of electron density presumably corresponding to Mg2+ is localized onto nsp16, distant from the SAH-binding cavity. The Mg2+ coordination mode is through six first-shell water molecules in an octahedral geometry (Fig. 4). Binding via water molecules, involves T58 and S188 side chain hydroxyls and the main chain carbonyl of E276. Since there are no carboxylic acids involved in binding this cation, it was suspected that its presence resulted from the crystallization procedure[42], with no biological relevance. However, the T58A, T58N, T58E and S188A substitutions show 43, 70, 99 and 72% loss of activity, respectively (Table 2), with no significant effect on the stability of the nsp10/nsp16 complex except for T58E whose association was 54 % that of wild-type. These residues are located on three distinct structural elements at the C-terminus of helix Z (T58), the N-terminus of β6 (S188), and in the central part of helix A3 (E276), respectively (Fig. 4 and Fig. S3). The cation may thus hold these elements together.


Crystal structure and functional analysis of the SARS-coronavirus RNA cap 2'-O-methyltransferase nsp10/nsp16 complex.

Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, Imbert I, Gluais L, Papageorgiou N, Sharff A, Bricogne G, Ortiz-Lombardia M, Lescar J, Canard B - PLoS Pathog. (2011)

Position and coordination of a metal ion.Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102710&req=5

ppat-1002059-g004: Position and coordination of a metal ion.Left, the putative Mg2+ ion (blue sphere) is shown solvated in its first atomic shell by six water molecules (red spheres). The corresponding 2Fo-Fc electron density map (contoured 1σ) with a cross shape is shown in purple. Residues of Nsp16 involved in coordination of Mg2+ ion via water molecules are labeled. Right, a global view of the bound metal ion in its electron density on the opposite side from the SAH molecule (red sticks). Nsp16 is shown as green ribbons, nsp10 as yellow ribbons with its two bound Zn2+ as grey spheres as in Fig. 1.
Mentions: We recently reported[16] that nsp10/nsp16 MTase activity requires Mg2+. Although the crystallization buffer contains Mg2+, we were unable to locate any such cation in the nsp16 active site. In enzyme activity assays, the Mg2+ ion can be substituted by Mn2+ or Ca2+, but not Zn2+ (data not shown, see also[16]). A peak of electron density presumably corresponding to Mg2+ is localized onto nsp16, distant from the SAH-binding cavity. The Mg2+ coordination mode is through six first-shell water molecules in an octahedral geometry (Fig. 4). Binding via water molecules, involves T58 and S188 side chain hydroxyls and the main chain carbonyl of E276. Since there are no carboxylic acids involved in binding this cation, it was suspected that its presence resulted from the crystallization procedure[42], with no biological relevance. However, the T58A, T58N, T58E and S188A substitutions show 43, 70, 99 and 72% loss of activity, respectively (Table 2), with no significant effect on the stability of the nsp10/nsp16 complex except for T58E whose association was 54 % that of wild-type. These residues are located on three distinct structural elements at the C-terminus of helix Z (T58), the N-terminus of β6 (S188), and in the central part of helix A3 (E276), respectively (Fig. 4 and Fig. S3). The cation may thus hold these elements together.

Bottom Line: We report the nsp10/nsp16 complex structure at 2.0 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų 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.

View Article: PubMed Central - PubMed

Affiliation: Centre National de la Recherche Scientifique and Université de la Méditerranée, UMR 6098, Architecture et Fonction des Macromolécules Biologiques, Marseille, France. etienne.decroly@afmb.univ-mrs.fr

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 Å resolution, which shows nsp10 bound to nsp16 through a ∼930 Ų 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.

Show MeSH
Related in: MedlinePlus