PDBsum entry 4wtf

Transferase/RNA

PDB id: 4wtf

Contents

Protein chain

536 a.a.

DNA/RNA

Ligands

5GS

Metals

_MN ×3

_CL

Waters ×81

PDB id:

4wtf

Name:

Transferase/RNA

Title:

Crystal structure of hcv ns5b genotype 2a jfh-1 isolate with s15g e86q e87q c223h v321i mutations and delta8 beta hairpin loop deletion in complex with gs-639475, mn2+ and symmetrical primer template 5'- caaaauuu

Structure:

RNA primer template caaaauuu. Chain: t, p. Engineered: yes. RNA-directed RNA polymerase. Chain: a. Engineered: yes. Mutation: yes

Source:

Synthetic: yes. Synthetic construct. Organism_taxid: 32630. Hepatitis c virus jfh-1. Organism_taxid: 356411. Variant: s15g c223h v321i delta8. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.65Å R-factor: 0.189 R-free: 0.234

Authors:

T.E.Edwards,T.C.Appleby,M.E.Mcgrath

Key ref:

T.C.Appleby et al. (2015). Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase. Science, 347, 771-775. PubMed id: 25678663 DOI: 10.1126/science.1259210

Date:

30-Oct-14 Release date: 11-Feb-15

Protein chain

Q99IB8  (POLG_HCVJF) -  Genome polyprotein from Hepatitis C virus genotype 2a (isolate JFH-1)

Seq: 3033 a.a.

Struc: 536 a.a.*

* PDB and UniProt seqs differ at 8 residue positions (black crosses)

DNA/RNA chains

C-A-A-A-A-U-U-U 8 bases

A-A-A-U-U-U 6 bases

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.3.4.21.98 - hepacivirin.
• Reaction: Hydrolysis of four peptide bonds in the viral precursor polyprotein, commonly with Asp or Glu in the P6 position, Cys or Thr in P1 and Ser or Ala in P1'.
• Enzyme class 4: E.C.3.4.22.- - ?????
• Enzyme class 5: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 6: 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: 10.1126/science.1259210

Science 347:771-775 (2015)

PubMed id: 25678663

Viral replication. Structural basis for RNA replication by the hepatitis C virus polymerase.

T.C.Appleby, J.K.Perry, E.Murakami, O.Barauskas, J.Feng, A.Cho, D.Fox, D.R.Wetmore, M.E.McGrath, A.S.Ray, M.J.Sofia, S.Swaminathan, T.E.Edwards.

ABSTRACT

Nucleotide analog inhibitors have shown clinical success in the treatment of hepatitis C virus (HCV) infection, despite an incomplete mechanistic understanding of NS5B, the viral RNA-dependent RNA polymerase. Here we study the details of HCV RNA replication by determining crystal structures of stalled polymerase ternary complexes with enzymes, RNA templates, RNA primers, incoming nucleotides, and catalytic metal ions during both primed initiation and elongation of RNA synthesis. Our analysis revealed that highly conserved active-site residues in NS5B position the primer for in-line attack on the incoming nucleotide. A β loop and a C-terminal membrane-anchoring linker occlude the active-site cavity in the apo state, retract in the primed initiation assembly to enforce replication of the HCV genome from the 3' terminus, and vacate the active-site cavity during elongation. We investigated the incorporation of nucleotide analog inhibitors, including the clinically active metabolite formed by sofosbuvir, to elucidate key molecular interactions in the active site.