PDBsum entry 3kqh

Hydrolase/DNA

PDB id: 3kqh

Contents

Protein chains

436 a.a. *

DNA/RNA

Waters ×138

* Residue conservation analysis

PDB id:

3kqh

Name:

Hydrolase/DNA

Title:

Three conformational snapshots of the hepatitis c virus ns3 helicase reveal a ratchet translocation mechanism

Structure:

Serine protease/ntpase/helicase ns3. Chain: a, b. Synonym: core protein p21, capsid protein c, p21, core protein p19, envelope glycoprotein e1, gp32, gp35, envelope glycoprotein e2, ns1, gp68, gp70, p7, protease ns2-3, p23, serine protease/ntpase/helicase ns3, hepacivirin, ns3p, p70, non-structural protein 4a, ns4a, p8, non-structural protein 4b, ns4b, p27, non-structural protein 5a, ns5a, p56, RNA-directed RNA polymerase, ns5b, p68. Engineered: yes.

Source:

Hepatitis c virus. Hcv. Organism_taxid: 333284. Strain: con1. Gene: ns3. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes

Resolution:

2.40Å R-factor: 0.226 R-free: 0.258

Authors:

M.Gu,C.M.Rice

Key ref:

M.Gu and C.M.Rice (2010). Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc Natl Acad Sci U S A, 107, 521-528. PubMed id: 20080715 DOI: 10.1073/pnas.0913380107

Date:

17-Nov-09 Release date: 26-Jan-10

Protein chains

Q9WMX2  (POLG_HCVCO) -  Genome polyprotein from Hepatitis C virus genotype 1b (isolate Con1)

Seq: 3010 a.a.

Struc: 436 a.a.

DNA/RNA chains

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

A-A-A-A-A-A 6 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.3.4.22.- - ?????
• Enzyme class 4: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 5: 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: 10.1073/pnas.0913380107

Proc Natl Acad Sci U S A 107:521-528 (2010)

PubMed id: 20080715

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism.

M.Gu, C.M.Rice.

ABSTRACT

A virally encoded superfamily-2 (SF2) helicase (NS3h) is essential for the replication of hepatitis C virus, a leading cause of liver disease worldwide. Efforts to elucidate the function of NS3h and to develop inhibitors against it, however, have been hampered by limited understanding of its molecular mechanism. Here we show x-ray crystal structures for a set of NS3h complexes, including ground-state and transition-state ternary complexes captured with ATP mimics (ADP.BeF(3) and ). These structures provide, for the first time, three conformational snapshots demonstrating the molecular basis of action for a SF2 helicase. Upon nucleotide binding, overall domain rotation along with structural transitions in motif V and the bound DNA leads to the release of one base from the substrate base-stacking row and the loss of several interactions between NS3h and the 3' DNA segment. As nucleotide hydrolysis proceeds into the transition state, stretching of a "spring" helix and another overall conformational change couples rearrangement of the (d)NTPase active site to additional hydrogen-bonding between NS3h and DNA. Together with biochemistry, these results demonstrate a "ratchet" mechanism involved in the unidirectional translocation and define the step size of NS3h as one base per nucleotide hydrolysis cycle. These findings suggest feasible strategies for developing specific inhibitors to block the action of this attractive, yet largely unexplored drug target.

Selected figure(s)

Figure 1.
Crystal Structures of NS3h Complexes. (A) NS3h in complex with ssDNA (dA[6]). (B) NS3h in complex with ADP·BeF[3] and ssDNA (dT[12], with five deoxynucleosides presented for clarity). (C) NS3h in complex with and ssDNA (dT[6]). The structures are represented by ribbons and transparent surfaces. The DNA bases and deoxyribose groups are shown with sticks and numerically labeled. The DNA phosphodiester backbones are simplified as orange tubes. The DNA atoms are color coded according to elements. The helicase motifs are color coded in the surface and ribbon respectively in (B) and (C). The distances between the Cα atoms of K244 (domain 1) and S403 (domain 2) are noted. ADP·BeF[3] and are shown with sticks and color coded.

Figure 6.
Schematic Presentation of Helicase Motion. (A) Schematic view of conformational changes between NS3h subdomains. The NS3h complexes are simplified as spherical modules (NS3h) and black lines (ssDNA). The two DNA-binding surfaces (NABS1 and NABS2) are noted. The yellow dots represent the sites involved in the coordination of phosphate groups of ssDNA. The W501 side chain is simplified as a black line. Nucleotides are noted as red letters. (B) Schematic view of ssDNA in the substrate-binding groove. Individual DNA residues are presented. The deoxyribose groups in C2′-endo pucker are labeled B, whereas the others in C3′-endo pucker are labeled A. The solid-black DNA bases are in syn orientation. The two DNA-binding surfaces are simplified as blue and pink modules. The black dashed lines represent hydrogen bonds between NS3h and the phosphodiester backbone of DNA, whereas the gray dashed lines are water-mediated interactions. (C) Fluorescence anisotropy titration in the absence and presence of ATP mimics. Data were fit to a quadratic equation to obtain dissociation constants (Kd).

Figures were selected by an automated process.