Crystal Structures of the RNA-dependent RNA Polymerase Genotype 2a of Hepatitis C Virus Reveal Two Conformations and Suggest Mechanisms of Inhibition by Non-nucleoside Inhibitors

Bichitra K Biswal,Maia M Cherney,Meitian Wang,Laval Chan,Constantin G Yannopoulos,Darius Bilimoria,Olivier Nicolas,Jean Bedard,Michael N.G James

doi:10.1074/jbc.m413410200

Abstract

Crystal structures of the RNA-dependent RNA polymerase genotype 2a of hepatitis C virus (HCV) from two crystal forms have been determined. Similar to the three-dimensional structures of HCV polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains. The main structural differences between the molecules in the two crystal forms lie at the interface of the fingers and thumb domains. The relative orientation of the thumb domain with respect to the fingers and palm domains and the beta-flap region is altered. Structural analysis reveals that the NS5B polymerase in crystal form I adopts a "closed" conformation that is believed to be the active form, whereas NS5B in crystal form II adopts an "open" conformation and is thus in the inactive form. In addition, we have determined the structures of two NS5B polymerase/non-nucleoside inhibitor complexes. Both inhibitors bind at a common binding site, which is nearly 35 A away from the polymerase active site and is located in the thumb domain. The binding pocket is predominantly hydrophobic in nature, and the enzyme inhibitor complexes are stabilized by hydrogen bonding and van der Waals interactions. Inhibitors can only be soaked in crystal form I and not in form II; examination of the enzyme-inhibitor complex reveals that the enzyme has undergone a dramatic conformational change from the form I (active) complex to the form II (inactive).

Highlights

MATERIALS AND METHODSPurification and Biochemical Studies—For crystallographic studies and measurement of the inhibitory effects of compounds, a soluble C-terminal truncated form of the polymerase 2a enzyme was obtained by the following approach
Hepatitis C virus (HCV)[1] is a debilitating human pathogen affecting an estimated 3% of the world’s population (1)
Similar to the three-dimensional structures of hepatitis C virus (HCV) polymerase genotype 1b and other known polymerases, the structures of the HCV polymerase genotype 2a in both crystal forms can be depicted in the classical right-hand arrangement with fingers, palm, and thumb domains

Summary

MATERIALS AND METHODS

Purification and Biochemical Studies—For crystallographic studies and measurement of the inhibitory effects of compounds, a soluble C-terminal truncated form of the polymerase 2a enzyme was obtained by the following approach. A second round of PCR (40 cycles at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 120 s) was performed by diluting the annealed fragments 100-fold and using the outer primers to create the complete full-length gene. Intensity data from both crystal forms and from the protein-inhibitor B complex were collected at the beam line 8.3.1 of the Advanced Light Source in Berkeley, CA, whereas data from the protein-inhibitor A complex were collected using an R-AXIS IV ϩϩ image plate detector with copper K␣ radiation generated by a Rigaku RU-300 rotating anode x-ray generator Both crystal forms contain one NS5B molecule in the asymmetric unit with solvent contents of ϳ64 and 60% in form I and form II, respectively (26). Structure Determination and Refinement—Structure solutions of both crystal forms were achieved by molecular replacement with the CNS package (28) using the unliganded 1b genotype polymerase structure (8) (Protein Data Bank code 1C2P) as the search model. Extensive model building was done in Structure of HCV RNA-dependent RNA Polymerase Genotype 2a

Complex B

RESULTS AND DISCUSSION

TABLE IV Structures used in the analysis

PDB code