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

Abstract 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.