Recognition of Divergent Viral Substrates by the SARS-CoV-2 Main Protease

Abstract

The main protease (M^pro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of coronavirus disease (COVID-19), is an ideal target for pharmaceutical inhibition. M^pro is conserved among coronaviruses and distinct from human proteases. Viral replication depends on the cleavage of the viral polyprotein at multiple sites. We present crystal structures of SARS-CoV-2 M^pro bound to two viral substrate peptides. The structures show how M^pro recognizes distinct substrates and how subtle changes in substrate accommodation can drive large changes in catalytic efficiency. One peptide, constituting the junction between viral nonstructural proteins 8 and 9 (nsp8/9), has P1' and P2' residues that are unique among the SARS-CoV-2 M^pro cleavage sites but conserved among homologous junctions in coronaviruses. M^pro cleaves nsp8/9 inefficiently, and amino acid substitutions at P1' or P2' can enhance catalysis. Visualization of M^pro with intact substrates provides new templates for antiviral drug design and suggests that the coronavirus lifecycle selects for finely tuned substrate-dependent catalytic parameters.

Keywords: Mpro; SARS-CoV-2; protease; virology.

Figure 1.

Viral M^pro substrates. (A) Protein sequence alignment of the 11 SARS-CoV-2 M^pro cleavage sites required for maturation of SARS-CoV2. (B) Protein sequence alignment of nsp8/9 M^pro cleavage sites from representative coronaviruses.

Figure 2.

Differential recognition of nsp4/5 and nsp8/9 substrates by M^pro. Identical views of nsp4/5 (A) and nsp8/9 (B) substrates in the M^proCys145Ala active site. Substrate peptide P and P′ residues are labeled with colored numbers. Key M^pro residues mentioned in the text are labeled. Conserved hydrogen bonds enabling M^pro recognition of substrate mainchain and P1 Gln side chain atoms are shown as white dashed lines. Hydrogen bonds that differ between the complex with nsp4/5 and that with nsp8/9 are shown in green and magenta, respectively. M^pro Asn142 and Gln189 contact both substrate through bound water molecules, and the resulting networks of hydrogen bonds differ between the two substrates.

Figure 3.

Steric effects that influence substrate recognition and M^pro activity. Spheres show positions of atoms dictating shape complementarity between M^pro subsites and nsp4/5 (A), nsp8/9 (B), and nsp5 (C; acyl-enzyme intermediate, PDB 7KHP). Labels show M^pro subsites and the distance between M^pro Met49 and Asn142 (thioether to amide nitrogen).