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. 2006 Aug 8;103(32):11892-7.
doi: 10.1073/pnas.0601708103. Epub 2006 Aug 1.

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family

Stefano Ricagno  1 , Marie-Pierre Egloff, Rachel Ulferts, Bruno Coutard, Didier Nurizzo, Valérie Campanacci, Christian Cambillau, John Ziebuhr, Bruno Canard
Affiliations

Crystal structure and mechanistic determinants of SARS coronavirus nonstructural protein 15 define an endoribonuclease family

Stefano Ricagno et al. Proc Natl Acad Sci U S A. .

Abstract

The approximately 30-kb coronavirus (+)RNA genome is replicated and transcribed by a membrane-bound replicase complex made up of 16 viral nonstructural proteins (nsp) with multiple enzymatic activities. The complex includes an RNA endonuclease, NendoU, that is conserved among nidoviruses but no other RNA virus, making it a genetic marker of this virus order. NendoU (nsp15) is a Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Neither biochemical nor sequence homology criteria allow a classification of nsp15 into existing endonuclease families. Here, we report the crystal structure of the severe acute respiratory syndrome coronavirus nsp15 at 2.6-A resolution. Nsp15 exhibits a unique fold and assembles into a toric hexamer with six potentially active, peripheric catalytic sites. The structure and the spatial arrangement of the catalytic residues into an RNase A-like active site define a separate endonuclease family, endoU, and represent another spectacular example of convergent evolution toward an enzymatic function that is critically involved in the coronavirus replication cycle.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Structure of nsp15, the endoribonuclease from SARS-CoV. (a and b) Cartoon representation (a) and topology diagram (b) of nsp15 monomer. The structure consists of three domains: N-terminal domain (α1–α2), central domain (α3–β8), and C-terminal domain (α6–β14). Secondary structures are colored as follows: red, α-helices; yellow, β-sheets; and green, loops. (c) A view of the nsp15 hexamer. In the trimer shown in the foreground, the subdomains of one of the monomers are colored as follows: green, N-terminal domain; yellow, central domain; and red, C-terminal domain. The other two molecules are shown in blue and magenta. The molecules belonging to the trimer in the background are shown in gray. (d) 90° rotation of the nsp15 hexamer as shown in c. (e) Surface representation of the nsp15 hexamer. Solid arrows indicate the active sites of the trimer in the foreground; dashed arrows show the active sites of the trimer in the background. (f) The hexamer shown in e is rotated by 90°. In this orientation, two active sites are visible, and they are highlighted by solid arrows and colored in white.
Fig. 2.
Fig. 2.
Sequence alignment of nsp15 from SARS-CoV and other coronaviruses as well as XendoU from X. laevis. The numbering refers to the SARS-CoV nsp15 sequence (SARS). nsp15 secondary structures are shown above the sequences. Several mutants of nsp15 have been previously reported (16, 17). Enzymes are shown with substitutions of residues highlighted in the following colors: blue, substitutions were affected in both their enzymatic activity and oligomerization state; pink, substitutions revealed enzymes with wild-type properties; green, substitutions affected the activity but not the oligomerization state. The four amino acids highlighted by an asterisk are the ones mutated in the present study. Fully conserved residues are shown in red. MHV, mouse hepatitis virus strain 2; BCoV, bovine coronavirus (strain 98TXSF-110-ENT); PEDV, porcine epidemic diarrhea virus (strain CV777); HV229E, HCoV-229E; AIBV, avian infectious bronchitis virus (strain Beaudette).
Fig. 3.
Fig. 3.
Catalytic site and proposed enzymatic mechanism. (a) Stereoview of the active site groove formed by the nsp15 C-terminal domain. The catalytic amino acids of bovine pancreatic RNase A (in cyan) are superimposed with the putative catalytic amino acids of nsp15 (in magenta). (b) Proposed enzymatic mechanism as suggested by the SARS-CoV nsp15 crystal structure and mutational studies, in analogy with the mechanism established for bovine pancreatic RNase A. (c) Stereoview of uridine 3′-phosphate (U3′MP) modeled in the nsp15 active site. The main hypothetical interactions between the catalytic amino acids and U3′MP are shown as dashed lines. (d) Stereoview of the electrostatic potential of the nsp15 active site groove. The modeled U3′MP molecule is shown in ball and stick representation.
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