Love R A, Parge H E, Wickersham J A, Hostomsky Z, Habuka N, Moomaw E W, Adachi T, Margosiak S, Dagostino E, Hostomska Z
Agouron Pharmaceuticals, Incorporated, San Diego, CA 92121, USA.
Clin Diagn Virol. 1998 Jul 15;10(2-3):151-6. doi: 10.1016/s0928-0197(98)00036-1.
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.
Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation.
We determined the crystal structure of NS3 proteinase of HCV BK isolate (genotype 1b; residues 1-189) and also the crystal structure of this proteinase complexed with HCV BK-NS4A (residues 21-34).
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 P1 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 alpha-helix and beta-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.
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 P1 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.
丙型肝炎病毒（HCV）非结构蛋白的释放需要NS3蛋白酶活性，因此它是一个潜在的抗病毒靶点。该酶需要位于多蛋白上NS3下游的蛋白辅因子NS4A来激活并进行有效加工。
比较NS4A结合前后蛋白酶的三维结构应有助于阐明NS4A依赖性酶激活的机制。
我们测定了HCV BK分离株（1b基因型；第1 - 189位氨基酸残基）的NS3蛋白酶的晶体结构，以及该蛋白酶与HCV BK - NS4A（第21 - 34位氨基酸残基）形成的复合物的晶体结构。
无辅因子（NS3P）或有结合辅因子（NS3P/4A）时，该酶的核心区域（第30 - 178位氨基酸残基）折叠成胰蛋白酶样构象，底物P1特异性口袋基本不变。然而，相对于NS3P，NS3P/4A中D1 - E1β环从辅因子结合位点移开，从而容纳NS4A。结果之一是催化残基His - 57和Asp - 81向Ser - 139靠近，且它们的侧链采取更“传统”（胰蛋白酶样）的取向。N端（第1 - 30位氨基酸残基）在NS3P中是伸展的，而在NS3P/4A中折叠成一个α螺旋和β链，覆盖结合的NS4A辅因子。由重新折叠的N端和NS4A形成了一个新的底物结合表面，可能影响切割位点下游紧邻的底物残基。
NS3P和NS3P/4A晶体结构的直接比较表明，NS4A的结合改善了酶催化三联体的锚定和取向。这与NS4A结合后NS3P微弱的残余活性增强是一致的。该酶的N端也有显著的重新折叠，这为与P'侧底物残基提供了新的相互作用。NS4A结合后，包括P1特异性口袋在内的P'侧底物残基的结合表面变化不大。总之，我们观察到NS3P与其NS4A辅因子形成复合物后底物周转和亲和力提高的结构基础。
