The S2 Pocket Governs the Genus‐Specific Substrate Selectivity of Coronavirus 3C‐Like Protease

Coronavirus 3C-like protease (CoV 3CL^pro) is essential for viral replication, providing an attractive target for monitoring the evolution of CoV and developing anti-CoV drugs. Here, the substrate-binding modes of 3CL^pros from four CoV genera are analyzed and found that the S2 pocket in 3CL^pro is highly conserved within each genus but differs between genera. Functionally, the S2 pocket, in conjunction with S4 and S1' pockets, governs the genus-specific substrate selectivity of 3CL^pro. Resurrected ancestral 3CL^pros from four CoV genera validate the genus-specific divergence of S2 pocket. Drawing upon the genus-specific S2 pocket as evolutionary marker, eight newly identified 3CL^pros uncover the ancestral state of modern 3CL^pro and elucidate the possible evolutionary process for CoV. It is also demonstrated that the S2 pocket is highly correlated with the genus-specific inhibitory potency of PF-07321332 (an FDA-approved drug against COVID-19) on different CoV 3CL^pros. This study on 3CL^pro provides novel insights to inform evolutionary mechanisms for CoV and develop genera-specific or broad-spectrum drugs against CoVs.