5ezs
Venezuelan Equine Encephalitis Virus (VEEV) Nonstructural Protein 2 (nsP2) Cysteine Protease Inhibited by E64d
Structural highlights
FunctionPOLN_EEVVT P123 and P123' are short-lived polyproteins, accumulating during early stage of infection. P123 is directly translated from the genome, whereas P123' is a product of the cleavage of P1234. They localize the viral replication complex to the cytoplasmic surface of modified endosomes and lysosomes. By interacting with nsP4, they start viral genome replication into antigenome. After these early events, P123 and P123' are cleaved sequentially into nsP1, nsP2 and nsP3/nsP3'. This sequence of delayed processing would allow correct assembly and membrane association of the RNA polymerase complex (By similarity). nsP1 is a cytoplasmic capping enzyme. This function is necessary since all viral RNAs are synthesized in the cytoplasm, and host capping enzymes are restricted to the nucleus. The enzymatic reaction involves a covalent link between 7-methyl-GMP and nsP1, whereas eukaryotic capping enzymes form a covalent complex only with GMP. nsP1 capping would consist in the following reactions: GTP is first methylated and then forms the m7GMp-nsP1 complex, from which 7-methyl-GMP complex is transferred to the mRNA to create the cap structure. Palmitoylated nsP1 is remodeling host cell cytoskeleton, and induces filopodium-like structure formation at the surface of the host cell (By similarity). nsP2 has two separate domain with different biological activities. The N-terminal section is part of the RNA polymerase complex and has RNA trisphosphatase and RNA helicase activity. The C-terminal section harbors a protease that specifically cleaves and releases the four mature proteins (By similarity). nsP3 and nsP3' are essential for minus strand and subgenomic 26S mRNA synthesis (By similarity). nsP4 is a RNA dependent RNA polymerase. It replicates genomic and antigenomic RNA by recognizing replications specific signals. Transcribes also a 26S subgenomic mRNA by initiating RNA synthesis internally on antigenomic RNA. This 26S mRNA encodes for structural proteins. nsP4 is a short-lived protein regulated by several ways: the opal codon readthrough and degradation by ubiquitin pathway (By similarity). Publication Abstract from PubMedThe 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 crystal structures of the free enzyme and the first inhibitor bound alphaviral protease structure. The peptide-like E64d inhibitor was found to bind beneath a beta-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, 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. A 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 Syndrome 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-fold to 400 microM. Within the beta-hairpin flap that clamps over the substrate, 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. Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease.,Hu X, Compton JR, Leary DH, Olson MA, Lee MS, Cheung J, Ye W, Ferrer M, Southall N, Jadhav A, Morazzani EM, Glass PJ, Marugan JJ, Legler PM Biochemistry. 2016 Mar 31. PMID:27030368[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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