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.


OBJECTIVE
Comparison of the proteinase three-dimensional structure before and after NS4A binding should help to elucidate the mechanism of NS4A-dependent enzyme activation.


METHODS
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).


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 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.


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 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.
Love
R A
RA
Agouron Pharmaceuticals, Incorporated, San Diego, CA 92121, USA. ral@agouron.com
Parge
H E
HE
Wickersham
J A
JA
Hostomsky
Z
Z
Habuka
N
N
Moomaw
E W
EW
Adachi
T
T
Margosiak
S
S
Dagostino
E
E
Hostomska
Z
Z
D001665:Binding Sites; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D016174:Hepacivirus / Q000737:chemistry* / Q000201:enzymology*; D006801:Humans; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D020365:RNA Helicases; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C080106:NS4A protein, flavivirus