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. 2007 Jun;81(12):6700-8.
doi: 10.1128/JVI.02817-06. Epub 2007 Apr 4.

Crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch

Jeremiah S Joseph  1 , Kumar Singh Saikatendu, Vanitha Subramanian, Benjamin W Neuman, Michael J Buchmeier, Raymond C Stevens, Peter Kuhn
Affiliations

Crystal structure of a monomeric form of severe acute respiratory syndrome coronavirus endonuclease nsp15 suggests a role for hexamerization as an allosteric switch

Jeremiah S Joseph et al. J Virol. 2007 Jun.

Abstract

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARS-CoV) contains a novel uridylate-specific Mn2+-dependent endoribonuclease (NendoU). Structure studies of the full-length form of the obligate hexameric enzyme from two CoVs, SARS-CoV and murine hepatitis virus, and its monomeric homologue, XendoU from Xenopus laevis, combined with mutagenesis studies have implicated several residues in enzymatic activity and the N-terminal domain as the major determinant of hexamerization. However, the tight link between hexamerization and enzyme activity in NendoUs has remained an enigma. Here, we report the structure of a trimmed, monomeric form of SARS-CoV nsp15 (residues 28 to 335) determined to a resolution of 2.9 A. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by approximately 120 degrees into the active site cleft. Furthermore, the catalytic nucleophile Lys 289 points in a diametrically opposite direction, a consequence of an outward displacement of the supporting loop (residues 276 to 295). In the full-length hexameric forms, these two loops are packed against each other and are stabilized by intimate intersubunit interactions. Our results support the hypothesis that absence of an adjacent monomer due to deletion of the hexamerization domain is the most likely cause for disruption of the active site, offering a structural basis for why only the hexameric form of this enzyme is active.

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Figures

FIG. 1.
FIG. 1.
Electrophoretic mobility shift assays. Monomeric SARS-CoV nsp15 (A and B) and native nsp10 (C and D) were incubated with fluoresceinated ssRNA substrate, and complexes were separated by native electrophoresis on 6% polyacrylamide gels. Fluoresceinated RNA was visualized under UV illumination (A and C), and protein was visualized similarly after SYPRO-ruby poststaining (B and D). Lanes: P, protein only; 0, RNA only; 1 to 7, gradient of protein with a fixed amount of RNA. Free RNA is indicated with a small filled triangle, protein is indicated with a large filled triangle, and protein-RNA complexes are indicated with open triangles. (E) nsp10 was used as a positive control; its binding affinity to the fluoresceinated RNA substrate is shown. The dotted lines indicate the concentration of nsp10 that correlates to 50% binding. (F) PFO-PAGE gel indicating that trimmed nsp15 exists primarily as a monomer and as a dimer; although a variety of larger forms exists, this construct appears to be specifically defective in trimer formation.
FIG. 2.
FIG. 2.
(a) Contents of the asymmetric unit. The four monomers (designated A, B, C, and D) are colored blue, green, cyan, and orange, respectively. (b) The three-domain topology of the truncated SARS-CoV nsp15 monomer. Color is ramped from the N terminus (blue) to the C terminus (red). Secondary structural elements are numbered β1 to β13 for β-strands, while α-helices are numbered α1 to α9. (c) Superimposition of the four monomers of the asymmetric unit. Subunits A/B, which form a pair, are colored blue and red, while the other two subunits, C and D, are colored green and magenta, respectively.
FIG. 3.
FIG. 3.
(a) Surface representation of hexameric full-length (FL) nsp15 (PDB code 2H85). The individual subunits are colored and marked A to F. The N-terminal 28 residues of the four monomers A, C, D, and F are shown in red and labeled AN, CN, DN, and FN, respectively. (b) Superposition of truncated (blue) and full-length (yellow) SARS-CoV nsp15 with MHV nsp15 (cyan). The genomic packaging signal of MHV is colored red, and the disordered region is shown as a dotted line. The sequence alignment of the packaging signal of MHV is shown below the ribbon diagram, with the implicated region (P192 - A215) indicated with a red line. Sequence IDs are gi:29837507 for SARS-CoV and gi:37999877 for MHV. (c) Conformational rearrangement of the active site loop and supporting loop in the truncated and full-length forms of nsp15. Truncated monomeric nsp15 is shown in green, while that of the hexameric WT is in light brown. The “supporting loop” (residues 276 to 295) and the “active site loop” (residues 234 to 249) are colored red in monomeric nsp15 and blue in full-length nsp15. Equivalent residues in the active sites of the two structures are labeled (regular font in monomeric nsp15 and in italics for the full-length enzyme). (d) Electron density map (2Fo-Fc) contoured at 1.1 σ around key residues in the active site of monomeric nsp15.
FIG. 3.
FIG. 3.
(a) Surface representation of hexameric full-length (FL) nsp15 (PDB code 2H85). The individual subunits are colored and marked A to F. The N-terminal 28 residues of the four monomers A, C, D, and F are shown in red and labeled AN, CN, DN, and FN, respectively. (b) Superposition of truncated (blue) and full-length (yellow) SARS-CoV nsp15 with MHV nsp15 (cyan). The genomic packaging signal of MHV is colored red, and the disordered region is shown as a dotted line. The sequence alignment of the packaging signal of MHV is shown below the ribbon diagram, with the implicated region (P192 - A215) indicated with a red line. Sequence IDs are gi:29837507 for SARS-CoV and gi:37999877 for MHV. (c) Conformational rearrangement of the active site loop and supporting loop in the truncated and full-length forms of nsp15. Truncated monomeric nsp15 is shown in green, while that of the hexameric WT is in light brown. The “supporting loop” (residues 276 to 295) and the “active site loop” (residues 234 to 249) are colored red in monomeric nsp15 and blue in full-length nsp15. Equivalent residues in the active sites of the two structures are labeled (regular font in monomeric nsp15 and in italics for the full-length enzyme). (d) Electron density map (2Fo-Fc) contoured at 1.1 σ around key residues in the active site of monomeric nsp15.
FIG. 4.
FIG. 4.
Superposition of XendoU structure (PDB ID 2C1W; green) onto the catalytic domain of SARS-CoV NendoU (PDB ID 2H85; light pink). The two disordered loop regions in the XendoU structure are indicated by dotted lines. The secondary structures are numbered using the same scheme as for Fig. 2b. The active site loops of the two structures are highlighted in red.
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