Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity

A comparison of the DNA sequences from all available genotypes of HCV indicate that the active site residues of the NS3 protease are strictly conserved with the exception of positions 123 and 168, which border the S(4) subsite. In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. To determine if these differences contribute to an altered specificity, we characterized single-chain NS3 proteases from strains 1a, 1b, and 3a with peptide substrates and product inhibitors on the basis of the natural cleavage junction sequences, in addition to polyprotein substrates derived from the 1a strain. No statistically significant differences in specificity were observed. To demonstrate that the active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed that there are measurable differences between the NS3 proteases of genotypes 1 and 3. Specifically, a 5-fold difference in K(i) was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P(5), and a 30-fold difference was seen when this position contained a D-homoglutamate. The contribution of residues 123 and 168 toward the altered specificity was then evaluated individually by site-directed mutagenesis. These mutants showed that potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural substrate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observations. Proteins 2001;43:82-88.