6bcm | pdb_00006bcm
Structure of a Self-inhibited N475A variant of the Venezuelan Equine Encephalitis Virus (VEEV) nsP2 cysteine protease
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 alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated anti-viral 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 where 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 the kcat/Km. A side-chain to substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in the 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. Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease leads to a Self-inhibited state.,Compton J, Mickey M, Hu X, Marugan JJ, Legler PM Biochemistry. 2017 Oct 24. doi: 10.1021/acs.biochem.7b00746. PMID:29064679[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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