Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease

Abstract

The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-L-methionine dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, 475NVCWAK480, which differs from papain’s (CGS25CWAFS), and the enzyme lacks a transition state (TS) stabilizing residue homologous to Q19 in papain. To understand the roles of conserved residues in catalysis we determined the structure of the free enzyme, and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a β-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme, each conformer may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 μM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold reductions in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle Eastern Respiratory virus (MERS). Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased the Km 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly, but not significantly increased the kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.

Design and Validation of Novel Chikungunya Virus Protease Inhibitors

“…These compounds were verified using cell culture assays, in which the most active of them had EC 90 s of 8.76 to 9.57 g/ml (33). For the protease of VEEV, a crystal structure of the enzyme with such an inhibitor has been recently resolved, clearly confirming the targeting of nsP2 and demonstrating the mode of binding of peptidomimetic inhibitors (34).…”

“…These findings once again highlight excellent correlation between predicted and experimentally revealed properties of the compounds used in this study. Furthermore, conformation-specific inhibition of protease activity demonstrates the high specificity of the inhibitor and also the high quality of the recombinant protein, opening the possibility to cocrystallize the enzyme with its inhibitor, as was recently done using a peptidomimetic inhibitor of VEEV protease (34). Such structures may further facilitate development of CHIKV nsP2 protease inhibitors as well as provide valuable information about the properties and functions of nsP2.…”

Chikungunya virus infectivity, RNA replication and non-structural polyprotein processing depend on the nsP2 protease’s active site cysteine residue

“…Subsequent molecular modelling revealed the features of the catalytic site and the S1′–S4 subsites22. A peptidomimetic inhibitor of nsP2 protease binds at the interface of the protease and MTL domains resulting in conformational change that most probably assists in leaving group departure of either the amine or Cys thiolate during the catalytic cycle23; the binding mode of natural substrates is most likely similar. In addition to the catalytic Cys, Asn475 and Lys480 residues (correspond to residues 476 and 481 of CHIKV nsP2) have been shown to be important for the protease activity of VEEV nsP223.…”

“…A peptidomimetic inhibitor of nsP2 protease binds at the interface of the protease and MTL domains resulting in conformational change that most probably assists in leaving group departure of either the amine or Cys thiolate during the catalytic cycle23; the binding mode of natural substrates is most likely similar. In addition to the catalytic Cys, Asn475 and Lys480 residues (correspond to residues 476 and 481 of CHIKV nsP2) have been shown to be important for the protease activity of VEEV nsP223. Known 3D structure coupled with the functional importance of nsP2 have made this protein an attractive target for the development of inhibitors of alphavirus infection232425262728.…”

“…In addition to the catalytic Cys, Asn475 and Lys480 residues (correspond to residues 476 and 481 of CHIKV nsP2) have been shown to be important for the protease activity of VEEV nsP223. Known 3D structure coupled with the functional importance of nsP2 have made this protein an attractive target for the development of inhibitors of alphavirus infection232425262728.…”