PDBsum entry 2f55

Hydrolase/DNA

PDB id: 2f55

Contents

Protein chains

432 a.a. *

DNA/RNA

Ligands

SO4 ×2

Waters ×62

* Residue conservation analysis

PDB id:

2f55

Name:

Hydrolase/DNA

Title:

Two hepatitis c virus ns3 helicase domains complexed with the same strand of DNA

Structure:

5'-d(p (Du)p (Du)p (Du)p (Du)p (Du)p (Du)p (Du)p (Du) p (Du)p (Du)p (Du)p (Du)p (Du))-3'. Chain: d. Engineered: yes. 5'-d(p (Du)p (Du)p (Du))-3'. Chain: e. Engineered: yes. Polyprotein. Chain: a, b, c.

Source:

Synthetic: yes. Hepatitis c virus. Organism_taxid: 11103. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Biol. unit:

Trimer (from PQS)

Resolution:

3.30Å R-factor: 0.247 R-free: 0.273

Authors:

J.Z.Lu,J.B.Jordan,J.Sakon

Key ref:

S.G.Mackintosh et al. (2006). Structural and biological identification of residues on the surface of NS3 helicase required for optimal replication of the hepatitis C virus. J Biol Chem, 281, 3528-3535. PubMed id: 16306038 DOI: 10.1074/jbc.M512100200

Date:

25-Nov-05 Release date: 06-Dec-05

Protein chains

O92972  (POLG_HCVJ4) -  Genome polyprotein from Hepatitis C virus genotype 1b (strain HC-J4)

Seq: 3010 a.a.

Struc: 432 a.a.

DNA/RNA chains

U-U-U-U-U-U-U-U-U-U-U-U-U 13 bases

U-U-U 3 bases

Enzyme reactions

• Enzyme class 1: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 2: 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 3: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 4: 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: 10.1074/jbc.M512100200

J Biol Chem 281:3528-3535 (2006)

PubMed id: 16306038

Structural and biological identification of residues on the surface of NS3 helicase required for optimal replication of the hepatitis C virus.

S.G.Mackintosh, J.Z.Lu, J.B.Jordan, M.K.Harrison, B.Sikora, S.D.Sharma, C.E.Cameron, K.D.Raney, J.Sakon.

ABSTRACT

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a protein-protein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only approximately 2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication.

Selected figure(s)

Figure 1.
X-ray crystal structure of NS3h bound to a 16-mer poly(dU) substrate. Three molecules of NS3h are found in the asymmetric unit, and two of the helicase monomers, chain A (red) and chain B (blue), are bound to a single DNA substrate molecule (aqua). The third molecule of the asymmetric unit (chain C, shown in green) is bound to a separate strand of nucleic acid. The DNA passes across the face of chain A, emerging between domains 2 and 3, where it enters the binding site of chain B.

Figure 3.
A, the terminal region of the oligonucleotide (aqua) interacts with NS3h chain A (red) via a hydrogen bond between Thr^269 (T269) and the phosphate backbone and ring stacking between nucleotide dU[1] and Trp^501 (W501; green). B, at the point where the oligonucleotide (aqua) enters the binding groove of chain B (blue), interactions identical to those with chain A are observed (Trp^501-dU ring stacking and Thr^269-phosphate hydrogen bond formation).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2006, 281, 3528-3535) copyright 2006.

Figures were selected by an automated process.