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SARS-CoV 3CL protease cleaves its C-terminal autoprocessing site by novel subsite cooperativity

Significance The SARS-CoV protease (3CLpro) has “noncanonical” substrate specificity for its C-terminal autoprocessing. Phe is required at both the second position upstream of the cleavage point (P2) and the third downstream position (P3′). This finding is surprising, given that 3CLpro reportedly requires Leu at the P2 position with no preference at the P3′ position. The conventional “consensus sequence” cannot explain this noncanonical specificity. Crystallography revealed that Phe at the P2 position changes the conformation of the substrate-binding pocket, and thereby creates the subsite for Phe at the P3′ position. This noncanonical specificity avoids the autoinhibition due to the mature C-terminal sequence of 3CLpro, which should be serious if Leu exists at the P2 position. The 3C-like protease (3CLpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) cleaves 11 sites in the polyproteins, including its own N- and C-terminal autoprocessing sites, by recognizing P4–P1 and P1′. In this study, we determined the crystal structure of 3CLpro with the C-terminal prosequence and the catalytic-site C145A mutation, in which the enzyme binds the C-terminal prosequence of another molecule. Surprisingly, Phe at the P3′ position [Phe(P3′)] is snugly accommodated in the S3′ pocket. Mutations of Phe(P3′) impaired the C-terminal autoprocessing, but did not affect N-terminal autoprocessing. This difference was ascribed to the P2 residue, Phe(P2) and Leu(P2), in the C- and N-terminal sites, as follows. The S3′ subsite is formed by Phe(P2)-induced conformational changes of 3CLpro and the direct involvement of Phe(P2) itself. In contrast, the N-terminal prosequence with Leu(P2) does not cause such conformational changes for the S3′ subsite formation. In fact, the mutation of Phe(P2) to Leu in the C-terminal autoprocessing site abolishes the dependence on Phe(P3′). These mechanisms explain why Phe is required at the P3' position when the P2 position is occupied by Phe rather than Leu, which reveals a type of subsite cooperativity. Moreover, the peptide consisting of P4–P1 with Leu(P2) inhibits protease activity, whereas that with Phe(P2) exhibits a much smaller inhibitory effect, because Phe(P3′) is missing. Thus, this subsite cooperativity likely exists to avoid the autoinhibition of the enzyme by its mature C-terminal sequence, and to retain the efficient C-terminal autoprocessing by the use of Phe(P2).