Multiple determinants influence complex formation of the hepatitis C virusNS3 protease domain with ils NS4A cofactor peptide

The interaction of the hepatitis C virus (HCV) NS3 protease domain with its NS4A cofactor peptide (Pep4AK) was investigated at equilibrium and at pre- steady state under different physicochemical conditions. Equilibrium dissoc iation constants of the NS3-Pep4AK complex varied by several orders of magn itude depending on buffer additives. Glycerol, NaCl, detergents, and peptid e substrates were found to stabilize this interaction. The extent of glycer ol-induced stabilization varied in an HCV strain-dependent way with at leas t one determinant mapping to an NS3-NS4A interaction site. Conformational t ransitions affecting at least the first 18 amino acids of NS3 were the main energy barriers for both the association and the dissociation reactions of the complex. However, deletion of this N-terminal portion of the protease molecule only slightly influenced equilibrium dissociation constants determ ined under different physicochemical conditions. Limited proteolysis experi ments coupled with mass spectrometric identification of cleavage fragments suggested a high degree of conformational flexibility affecting at least th e first 21 residues of NS3, The accessibility of this region of the proteas e to limited chymotryptic digestion did not significantly change in any con dition tested, whereas a significant reduction of chymotryptic cleavages wi thin the NS3 core was detected under conditions of high NS3-Pep4AK complex affinity. We conclude the following: (1) The N-terminus of the NS3 protease that, according to the X-ray crystal structure, makes extensive contacts w ith the cofactor peptide is highly flexible in solution and contributes onl y marginally to the thermodynamic stability of the complex. (2) Affinity en hancement is accomplished by several factors through a general stabilizatio n of the fold of the NS3 molecule.