Crystal structure of nonstructural protein 10 from the severe acute respiratory syndrome coronavirus reveals a novel fold with two zinc-binding motifs

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) possesses a large 29.7-kb positive-stranded RNA genome. The first open reading frame encodes replicase polyproteins 1a and 1ab, which are cleaved to generate 16 "nonstructural" proteins, nsp1 to nsp16, involved in viral replication and/or RNA processing. Among these, nsp10 plays a critical role in minus-strand RNA synthesis in a related coronavirus, murine hepatitis virus. Here, we report the crystal structure of SARS-CoV nsp10 at a resolution of 1.8 A as determined by single-wavelength anomalous dispersion using phases derived from hexatantalum dodecabromide. nsp10 is a single domain protein consisting of a pair of antiparallel N-terminal helices stacked against an irregular beta-sheet, a coil-rich C terminus, and two Zn fingers. nsp10 represents a novel fold and is the first structural representative of this family of Zn finger proteins found so far exclusively in coronaviruses. The first Zn finger coordinates a Zn2+ ion in a unique conformation. The second Zn finger, with four cysteines, is a distant member of the "gag-knuckle fold group" of Zn2+-binding domains and appears to maintain the structural integrity of the C-terminal tail. A distinct clustering of basic residues on the protein surface suggests a nucleic acid-binding function. Gel shift assays indicate that in isolation, nsp10 binds single- and double-stranded RNA and DNA with high-micromolar affinity and without obvious sequence specificity. It is possible that nsp10 functions within a larger RNA-binding protein complex. However, its exact role within the replicase complex is still not clear.

FIG. 1.

Sequence of SARS-CoV nsp10. Shown is a schematic of the SARS-CoV genome depicting the location of the replicase polyprotein and the different structural and accessory genes. The immediate neighborhood of nsp10 within pp1a/1ab is expanded in the inset, under which a sequence alignment of different homologues of nsp10 in coronaviruses is shown. RdRp, RNA-dependent RNA polymerase; TGEV, porcine transmissible gastroenteritis virus; IBV, avian infectious bronchitis virus; Hel, helicase; ExoN, exonuclease; Nendoll, endonuclease; 2′-o-MT, 2′-o-methyltransferase; EDV, epidemic diarrhea virus.

FIG. 2.

Structure of SARS-CoV nsp10. (a) Ribbon diagram of SARS-CoV nsp10 showing the arrangement of helices and strands. The secondary structures are colored from blue (N terminus) to red (C terminus) and are numbered from H1 to H5 for helices and 1 to 5 for the β-strands. (b) Topology diagram showing the connectivities between the secondary structural elements in the nsp10 structure. Helices are in cyan and strands are in yellow, with the same numbering scheme as that described for panel a. (c) Electron density observed at the first Zn²⁺-binding site. The residues coordinating the Zn²⁺ ion are shown as balls and sticks. (d) Electron density observed at the second Zn²⁺-binding site. The four cysteine residues coordinating the metal ion at the second Zn²⁺ ion near the protein C terminus are shown as balls and sticks. The 2F_o-F_c maps are contoured at 1.0 σ, where F_o and F_c are the observed and calculated structure factors, respectively.

FIG. 3.

Analysis of electrostatic charge distribution and symmetry mates of nsp10. (a) The surface of one of the nsp10 monomers is shown with electrostatic potential colored from blue (positive) to red (negative) in the range of +4.1 kT to −4.1 kT. Important residues that contribute to the positive charge are shown as balls and sticks over a semitransparent protein surface. The two metal ions are shown as spheres. (b) Two symmetry-related dimers observed in the crystal. The two zinc atoms Zn1 and Zn2 are shown. (c) Modeled effect of a Q65E mutation on the surface charge of nsp10. A large contiguous negatively charged surface patch is generated which may unfavorably alter a critical binding interface with an interacting protein and/or RNA. This mutation causes a temperature-sensitive defect in minus-strand RNA synthesis in MHV (25). WT, wild type.

FIG. 4.

Nucleic acid binding by nsp10. Binding mixtures containing (A) 80 pmol double-stranded RNA, (B) 82 pmol double-stranded DNA, (C) 100 pmol single-stranded RNA, or (D) no nucleic acid were incubated with various concentrations of nsp10. Binding mixtures shown in lanes 0 to 7 contained 0, 10, 20, 40, 80, 160, 320, and 800 μM nsp10, respectively. Free and bound nucleic acids were detected by SYBR-gold staining (Invitrogen). A double-stranded DNA ladder (1 Kb Plus; Invitrogen) was included as a marker (lane M). The positions of free nucleic acid (F), shifted nucleic acid-nsp10 complexes (S), and supershifted multiplexes (O) on the gels are indicated at the left. The shifted and supershifted nucleic acid peaks coincide with SYPRO-ruby protein stain in each gel (not shown).