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

The study investigates the role of the Asn-475 residue in the alpha viral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV). Mutating this residue led to an unexpected self-inhibited state, suggesting its importance in stabilizing and orienting essential elements during catalytic reactions.

Context and Aim of the Study

• The research was directed towards understanding the enzyme behavior of the alphaviral nsP2 cysteine protease in the VEEV, recognized as a potential target for antiviral drugs.
• Clan CN proteases, such as this one, possess both a cysteine protease domain for breakage of peptide bonds, and an RNA methyltransferase (SAM MTase) domain responsible for transferring a methyl group to RNA molecules.
• The study focused on the NVCWAK motif which includes the Nucleophilic cysteine, distinguishing the protease from the one found in papain.
• The aim was to explore the role of the Asn-475 residue in this motif through site-directed mutagenesis involving replacement of Asn-475 with alanine (N475A).

Findings and Implications

• The mutation to alanine resulted in a significant change in behaviour of the enzyme leading to an inactive or ‘self-inhibited’ state, wherein the N-terminus residues folded into the substrate binding cleft, inducing a conformational change. These residues were not hydrolyzed, indicating a catalytically incompetent state.
• The N475A mutation led to a substantial decrease (70-fold) in the rate constant for a reaction (k/K).
• Another result was the predicted interaction between the side-chain and substrate based on the structure. Further mutation (S701A) resulted in a 17-fold increase in the rate constant K.
• The study observed that Asn is also present in similar positions in other proteases, such as coronaviral papain-like proteases and human ubiquitin-specific proteases, suggesting its importance across similar enzymes.
• The findings highlighted that the Asn-475 residue holds significance in stabilizing the N-terminal residues and orienting the carbonyl group during nucleophilic attack. However, it was noted that it does not directly hydrogen bond the oxyanion.
• The results indicate that the self-inhibited state captured during this study provides valuable insights for designing peptide-like inhibitors and designing future antiviral drugs.