THE CONFORMATION OF HEPATITIS-C VIRUS NS3 PROTEINASE WITH AND WITHOUTNS4A - A STRUCTURAL BASIS FOR THE ACTIVATION OF THE ENZYME BY ITS COFACTOR

Background: Hepatitis C virus (HCV) NS3 proteinase activity is require d for the release of HCV nonstructural proteins and is thus a potentia l antiviral target. The enzyme requires a protein cofactor NS4A, locat ed downstream of NS3 on the polyprotein, for activation and efficient processing. Objectives: Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation. Study design: We determ ined the crystal structure of NS3 proteinase of HCV BK isolate (genoty pe Ib; residues 1-189) and also the crystal structure of this proteina se complexed with HCV BK-NS4A (residues 21-34). Results: The core regi on (residues 30-178) of the enzyme without cofactor (NS3P) or with bou nd cofactor (NS3P/4A) is folded into a trypsin-like conformation and t he substrate PI specificity pocket is essentially unchanged. However, the D1-E1 beta-loop shifts away from the cofactor binding site in NS3P /4A relative to NS3P, thereby accommodating NS4A. One result is that c atalytic residues His-57 and Asp-81 move closer to Ser-139 and their s idechains adopt more 'traditional' (trypsin-like) orientation. The N-t erminus (residues 1-30), while extended in NS3P, is folded into an a-h elix and P-strand that cover the bound cofactor of NS3P/4A. A new subs trate-binding surface is formed from both the refolded N-terminus and NS4A, potentially affecting substrate residues immediately downstream of the cleavage site. Conclusions: Direct comparison of the crystal st ructures of NS3P and NS3P/4A shows that the binding of NS4A improves t he anchoring and orientation of the enzyme's catalytic triad. This is consistent with the enhancement of NS3P's weak residual activity upon NS4A binding. There is also significant refolding of the enzyme's N-te rminus which provides new interactions with P'-side substrate residues . The binding surface for P'-side substrate residues, including the Pi specificity pocket, changes little after NS4A binding. In summary, we observe a structural basis for improved substrate turnover and affini ty that follows complexation of NS3P with its NS4A cofactor. (C) 1998 Elsevier Science B.V. All rights reserved.