Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.

Paradigms and Technologies
Therapeutic Approaches


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 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.


Compton, Jaimee R; Mickey, Matthew J; Hu, Xin; Marugan, Juan; Legler, Patricia M;


  • Amino Acid Sequence
  • Binding Sites
  • Binding, Competitive
  • Catalytic Domain
  • Crystallography, X-Ray
  • Cysteine Endopeptidases/ chemistry
  • Cysteine Endopeptidases/ genetics
  • Cysteine Endopeptidases/ metabolism
  • Encephalitis Virus, Venezuelan Equine/ enzymology
  • Feedback, Physiological
  • Humans
  • Hydrolysis
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutation
  • Protein Conformation
  • Sequence Homology
  • Viral Proteins/ chemistry
  • Viral Proteins/ genetics
  • Viral Proteins/ metabolism

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