Crystal Structure of a Monomeric Form of Severe Acute Respiratory Syndrome Coronavirus Endonuclease nsp15 Suggests a Role for Hexamerization as an Allosteric Switch

Abstract

Mature nonstructural protein-15 (nsp15) from the severe acute respiratory syndrome coronavirus (SARSCoV) contains a novel uridylate-specific Mn 2؉ -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 Å. The catalytic loop (residues 234 to 249) with its two reactive histidines (His 234 and His 249) is dramatically flipped by ϳ120°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.

Nidovirus ribonucleases: Structures and functions in viral replication

“…77,82 In the structure of a truncated, monomeric form of SARS-CoV nsp15 that lacked 28 N-terminal and 11 C-terminal residues, two loops of the catalytic domain (residues 234 to 249 and 276 to 295) were found to be significantly displaced compared to their location in the hexamer, resulting in the destruction of the active site. 87 In the hexamer, these two loops pack against each other and are stabilized in their position by inter-monomer interactions, suggesting a role for hexamerization as an allosteric switch. The interactions involved in positioning these loops are exclusively located at the trimerization interface.…”

SARS-CoV ORF1b-encoded nonstructural proteins 12–16: Replicative enzymes as antiviral targets

“…Moreover, expression of nsp15 in fusion with the maltose binding protein (MBP) still exhibits endoribonuclease activity even though the MBP prevents hexamer formation (Ivanov et al, 2004a). Finally, the structure of SARS-CoV nsp15 with deleted N-terminal region was determined, revealing that without its adjacent monomer in the hexameric ring, nsp15 cannot be active (Joseph et al, 2007).…”

Crystallization and preliminary X-ray crystallographic analysis of a nonstructural protein 15 mutant fromHuman coronavirus 229E

“…In previous investigations on nsp15s, even though several sitedirected mutations were made, trimers only existed in the transition state between hexamers and monomers. Stable trimeric nsp15 has not been obtained before; thus, its structure and enzymatic activity have not yet been reported (Ricagno et al, 2006;Joseph et al, 2007;Xu et al, 2006). In this work, trimeric nsp15 from HCoV-229E was successfully obtained by mutating Ile26 and Asn52 to alanine, and the expression, purification, crystallization and preliminary X-ray diffraction studies of this trimeric nsp15 are described.…”

“…In addition, the structure of the monomeric nsp15 in SARS-CoV was determined through an N-terminal domain truncation (PDB entry 2ozk; Joseph et al, 2007). Hexamers have been considered to be the fully active form, while monomers lose the enzymatic activity owing to a lack of RNAbinding ability (Bhardwaj et al, 2004;Joseph et al, 2007). In previous investigations on nsp15s, even though several sitedirected mutations were made, trimers only existed in the transition state between hexamers and monomers.…”

“…The crystal structures of these two homologous nsp15s have been determined previously (Xu et al, 2006;Ricagno et al, 2006), demonstrating that nsp15 is a hexamer formed by a dimer of trimers (PDB entries 2gth and 2h85). In addition, the structure of the monomeric nsp15 in SARS-CoV was determined through an N-terminal domain truncation (PDB entry 2ozk; Joseph et al, 2007). Hexamers have been considered to be the fully active form, while monomers lose the enzymatic activity owing to a lack of RNAbinding ability (Bhardwaj et al, 2004;Joseph et al, 2007).…”