6bcm

Publication Abstract from PubMed

The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated anti-viral 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 where 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 the kcat/Km. A side-chain to substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in the Km. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack, but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis, but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.

Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease leads to a Self-inhibited state.,Compton J, Mickey M, Hu X, Marugan JJ, Legler PM Biochemistry. 2017 Oct 24. doi: 10.1021/acs.biochem.7b00746. PMID:29064679^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.