PDBsum entry 5ezs

Hydrolase/hydrolase inhibitor

PDB id: 5ezs

Contents

Protein chain

320 a.a.

Ligands

E6D

Waters ×208

PDB id:

5ezs

Name:

Hydrolase/hydrolase inhibitor

Title:

Venezuelan equine encephalitis virus (veev) nonstructural protein 2 (nsp2) cysteine protease inhibited by e64d

Structure:

Non-structural protein 2 cysteine protease. Chain: a. Engineered: yes

Source:

Venezuelan equine encephalitis virus (strain trinidad donkey). Veev. Organism_taxid: 11038. Strain: trinidad donkey. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.16Å R-factor: 0.192 R-free: 0.227

Authors:

P.M.Legler

Key ref:

X.Hu et al. (2016). Kinetic, Mutational, and Structural Studies of the Venezuelan Equine Encephalitis Virus Nonstructural Protein 2 Cysteine Protease. Biochemistry, 55, 3007-3019. PubMed id: 27030368 DOI: 10.1021/acs.biochem.5b00992

Date:

26-Nov-15 Release date: 13-Apr-16

Protein chain

P27282  (POLN_EEVVT) -  Polyprotein P1234 from Venezuelan equine encephalitis virus (strain Trinidad donkey)

Seq: 2493 a.a.

Struc: 320 a.a.

Enzyme reactions

• Enzyme class 1: E.C.2.1.1.- - ?????
• Enzyme class 2: E.C.2.7.7.- - ?????
• Enzyme class 3: E.C.2.7.7.19 - polynucleotide adenylyltransferase.
• Reaction: RNA(n) + ATP = RNA(n)-3'-adenine ribonucleotide + diphosphate
• Enzyme class 4: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 5: E.C.3.1.3.84 - ADP-ribose 1''-phosphate phosphatase.
• Reaction: ADP-alpha-D-ribose 1''-phosphate + H2O = ADP-D-ribose + phosphate
• Enzyme class 6: E.C.3.4.22.- - ?????
• Enzyme class 7: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 8: E.C.3.6.1.74 - mRNA 5'-phosphatase.
• Reaction: a 5'-end triphospho-ribonucleoside in mRNA + H2O = a 5'-end diphospho- ribonucleoside in mRNA + phosphate + H⁺
• Enzyme class 9: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1021/acs.biochem.5b00992

Biochemistry 55:3007-3019 (2016)

PubMed id: 27030368

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

X.Hu, J.R.Compton, D.H.Leary, M.A.Olson, M.S.Lee, J.Cheung, W.Ye, M.Ferrer, N.Southall, A.Jadhav, E.M.Morazzani, P.J.Glass, J.Marugan, P.M.Legler.

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, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 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; one or both of the conformers 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 decreases 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 East Respiratory Syndrome (MERS) virus. 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 Km 4.5-fold to 500 μM. Within the β-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.