Refolding of lid subdomain of SARS-CoV-2 nsp14 upon nsp10 interaction releases exonuclease activity

Abstract

During RNA replication, coronaviruses require proofreading to maintain the integrity of their large genomes. Nsp14 associates with viral polymerase complex to excise the mismatched nucleotides. Aside from the exonuclease activity, nsp14 methyltransferase domain mediates cap methylation, facilitating translation initiation and protecting viral RNA from recognition by the innate immune sensors. The nsp14 exonuclease activity is modulated by a protein co-factor nsp10. While the nsp10/nsp14 complex structure is available, the mechanistic basis for nsp10-mediated modulation remains unclear in the absence of the nsp14 structure. Here, we provide a crystal structure of nsp14 in an apo-form. Comparative analysis of the apo- and nsp10-bound structures explain the modulatory role of the co-factor protein and reveal the allosteric nsp14 control mechanism essential for drug discovery. Further, the flexibility of the N-terminal lid of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp14 structure presented in this study rationalizes the recently proposed idea of nsp14/nsp10/nsp16 ternary complex.

Keywords: SAH; SARS-CoV-2; XRD; coronavirus; methylation; methyltransferase; nsp10; nsp14; proof-reading; structure.

Graphical abstract

Figure 1

Crystal structure of apo-nsp14 (A) Arrangement of the molecules in the asymmetric unit. Model (A) blue and model (B) pink. MTase and ExoN domains within the bimodular structure of nsp14 are distinguished by a primary color shade. Active-sites residues are indicated in red in one molecule, and the detailed arrangements of the active sites are shown in inserts. (B) Interactions of SAH (orange, stick model) at the active site of nsp14 MTase domain (pink, ribbon). Hydrogen bonds are depicted as dotted lines; waters are indicated as red spheres. See also Figure S1.

Figure 2

Nsp10 induced refolding of nsp14 ExoN lid subdomain (A) Nsp10 (navy blue, ribbon)/Nsp14 (gray, surface) complex overlaid on apo-nsp14 (cyan). The N-terminal lid of nsp14 refolds upon binding of nsp10 as exemplified by Thr50-Met58 region assuming a helical structure in apo-nsp14 and forming a strand of a β sheet indicated in nsp10/nsp14 complex. In the apo structure, the lid occludes nsp10-binding site at the surface of nsp14. (B) The RNA interaction site of nsp10 (navy blue)/nsp14 (beige) complex (7N0B) is sterically occluded by lid subdomain in apo-nsp14 structure (cyan), providing a rationale for the negligible exonuclease activity of nsp14 in the absence of nsp10. RNA (orange). The apo-nsp14 loop, occluding the RNA-binding site, is denoted in dark cyan (dotted line). See also Figures S2 and S3.