The conformation of hepatitis C virus NS3 proteinase with and without NS4A: a structural basis for the activation of the enzyme by its cofactor

Background: Hepatitis C virus (HCV) NS3 proteinase activity is required for the release of HCV nonstructural proteins and is thus a potential antiviral target. The enzyme requires a protein cofactor NS4A, located 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 determined the crystal structure of NS3 proteinase of HCV BK isolate (genotype Ib; residues 1-189) and also the crystal structure of this proteinase complexed with HCV BK-NS4A (residues 21-34). Results: The core region (residues 30-178) of the enzyme without cofactor (NS3P) or with bound cofactor (NS3P/4A) is folded into a trypsin-like conformation and the 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 catalytic residues His-57 and Asp-81 move closer to Ser-139 and their sidechains adopt more 'traditional' (trypsin-like) orientation. The N-terminus (residues 1-30), while extended in NS3P, is folded into an a-helix and P-strand that cover the bound cofactor of NS3P/4A. A new substrate-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 structures of NS3P and NS3P/4A shows that the binding of NS4A improves the 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-terminus 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 affinity that follows complexation of NS3P with its NS4A cofactor. (C) 1998 Elsevier Science B.V. All rights reserved.