Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease leads to a Self-inhibited state

The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, 475NVCWAK480, which differs from that of papain (22CGSCWAFS29). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state in which the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed—consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in kcat/Km. A side chain–substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in Km. An Asn at the n-2 position relative to the Cys ...