PDBsum entry 4zp6

Transferase

PDB id: 4zp6

Contents

Protein chain

454 a.a.

Ligands

SO4 ×4

Waters ×548

PDB id:

4zp6

Name:

Transferase

Title:

Coxsackievirus b3 polymerase - f364a mutant

Structure:

Genome polyprotein. Chain: a. Fragment: unp residues 1724-2185. Engineered: yes. Mutation: yes

Source:

Coxsackievirus b3. Organism_taxid: 12072. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.65Å R-factor: 0.190 R-free: 0.220

Authors:

O.B.Peersen,S.M.Mcdonald

Key ref:

S.McDonald et al. (2016). Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo. J Biol Chem, 291, 13999-14011. PubMed id: 27137934 DOI: 10.1074/jbc.M116.726596

Date:

07-May-15 Release date: 11-May-16

Protein chain

Q5UEA2  (Q5UEA2_9ENTO) -  Genome polyprotein from Coxsackievirus B3

Seq: 2185 a.a.

Struc: 454 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 3: E.C.3.4.22.28 - picornain 3C.
• Reaction: Selective cleavage of Gln-|-Gly bond in the poliovirus polyprotein. In other picornavirus reactions Glu may be substituted for Gln, and Ser or Thr for Gly.
• Enzyme class 4: E.C.3.4.22.29 - picornain 2A.
• Reaction: Selective cleavage of Tyr-|-Gly bond in the picornavirus polyprotein. In other picornavirus reactions Glu may be substituted for Gln, and Ser or Thr for Gly.
• Enzyme class 5: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + 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: 10.1074/jbc.M116.726596

J Biol Chem 291:13999-14011 (2016)

PubMed id: 27137934

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo.

S.McDonald, A.Block, S.Beaucourt, G.Moratorio, M.Vignuzzi, O.B.Peersen.

ABSTRACT

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.