6BCM

Structure of a Self-inhibited N475A variant of the Venezuelan Equine Encephalitis Virus (VEEV) nsP2 cysteine protease

Summary for 6BCM

• Entry DOI: 10.2210/pdb6bcm/pdb
• Descriptor: Protease nsP2, GLYCEROL, SODIUM ION, ... (4 entities in total)
• Functional Keywords: alphavirus, nsp2, nonstructural protein, cysteine protease, self-inhibited, n475a, s-adenosyl-l-methionine methyltransferase, viral protein
• Biological source: Venezuelan equine encephalitis virus
• Total number of polymer chains: 1
• Total formula weight: 38505.95

Authors

Compton, J.R.,Legler, P.M. (deposition date: 2017-10-20, release date: 2017-11-08, Last modification date: 2023-10-04)

Primary citation

Compton, J.R.,Mickey, M.J.,Hu, X.,Marugan, J.J.,Legler, P.M.
Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.
Biochemistry, 56:6221-6230, 2017

PubMed Abstract: 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, NVCWAK, which differs from that of papain (CGSCWAFS). 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 k/K. A side chain-substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in K. 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.

PubMed: 29064679
DOI: 10.1021/acs.biochem.7b00746

Experimental method

X-RAY DIFFRACTION (2.1 Å)