Structural basis of mismatch recognition by a SARS-CoV-2 proofreading enzyme

Fig. 1.. Structural and functional overview of SARS-CoV-2 nsp10-nsp14-RNA complexes. (A) Domain organization of SARS-CoV-2 nsp10 and nsp14. Domain boundary residues are numbered. Five catalytic residues in the nsp14 ExoN domain are indicated as red dots and are labeled. (B) Cleavage of T35P31 RNA substrate by SARS-CoV-2 nsp10-nsp14 ExoN complex. The nanomolar concentrations of WT or E191A mutant ExoN are indicated. The RNAs were resolved by denaturing polyacrylamide gel electrophoresis (PAGE) and stained by SYBR Gold. A representative result from three biological replicates is shown. (C) Sequence and numbering of the T35P31 RNA substrate used in biochemical characterization and structural determination. T-strand (template strand) and P-strand (product strand) RNAs are connected by a UUCG tetraloop. (D) Cryo-EM map and atomic model of the monomeric form of the SARS-CoV-2 nsp10-nsp14-RNA complex. (E) Cryo-EM map and atomic model of the tetrameric form of the SARS-CoV-2 nsp10-nsp14-RNA complex. A, Ala; D, Asp; E, Glu; H, His.

Fig. 2.. Active-site conformation and catalytic mechanism of SARS-CoV-2 ExoN. (A) Superimposition of the SARS-CoV nsp10-nsp14 complex (blue; PDB ID 5C8U) and the SARS-CoV-2 nsp10-nsp14-RNA complex (orange) illustrates the conformational changes of the α2-α3 and α4-α5 loops and a 2.6-Å shift of H268 toward the RNA upon substrate binding. (B) Active-site structure of the SARS-CoV-2 nsp10-nsp14 (WT)–RNA complex. Ca2+ ions, green spheres; catalytic water, red sphere. Nucleotide residues in P-strand RNA are indicated with a subscript “P.” +1CP, –1CP, the catalytic water, and two active-site metal ions are superimposed with their cryo-EM densities contoured at 10σ. (C) Active-site structure of the SARS-CoV-2 nsp10-nsp14 (E191A)–RNA complex. Mg2+ ions, green spheres. +1CP, –1CP, and two active-site metal ions are superimposed with their cryo-EM densities contoured at 7σ.

Fig. 3.. Mechanism of substrate recognition by SARS-CoV-2 ExoN. (A) Interactions between SARS-CoV-2 nsp10-nsp14 ExoN and the T35P31 RNA backbone. Nucleotide residues in T-strand RNA are indicated with a subscript “T,” whereas nucleotide residues in P-strand RNA are indicated with a subscript “P.” Hydrogen bonds and salt bridges are shown as gray dotted lines. Interacting nucleotide and protein residues are superimposed with their cryo-EM densities contoured at 7σ. (B) Interactions between SARS-CoV-2 nsp10-nsp14 ExoN and T35P31 RNA at +1 and –1 nucleobase positions. Hydrogen bonds and salt bridges are shown as gray dotted lines. π-π stacking interactions are indicated by green dotted lines. Interacting nucleotide and protein residues are superimposed with their cryo-EM densities contoured at 7σ. (C) Surface representation of the SARS-CoV-2 nsp10-nsp14 ExoN substrate binding pocket shows a restricted opening on the T-strand side that prevents base-pairing at the substrate RNA +1 position. For clarity, the N7-MTase domain of nsp14 is not shown. (D) Surface representation of the LASV NP ExoN domain. A fully base-paired dsRNA substrate (shown in cartoon representation) is bound in the…

Fig. 4.. Structural insights into antiviral design. (A) Interactions between SARS-CoV-2 nsp10-nsp14 ExoN and the 3′-end nucleotide mediated by its 2′- and 3′-OH groups. Mg2+ ion, green sphere. Hydrogen bonds are shown as gray dotted lines. +1CP and its interacting protein residues are superimposed with cryo-EM densities contoured at 7σ. (B) Cleavage of various P32 ssRNA substrates by the SARS-CoV-2 nsp10-nsp14 complex. The 3′-end CMP that bears different modifications at its 2′- or 3′-OH groups is shown as a black letter in a white circle. The nanomolar concentrations of ExoN are indicated. The RNAs were resolved by denaturing PAGE and stained by SYBR Gold. A representative result from three biological replicates is shown. (C) Predicted interactions between SARS-CoV-2 nsp10-nsp14 ExoN and RMP, which is modeled at the 3′-end +1 position of P-strand RNA. G, Gly.