Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its 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 dissociation constants of the NS3-Pep4AK complex varied by several orders of magnitude depending on buffer additives. Glycerol, NaCl, detergents, and peptide substrates were found to stabilize this interaction. The extent of glycerol-induced stabilization varied in an HCV strain-dependent way with at least one determinant mapping to an NS3-NS4A interaction site. Conformational transitions 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 determined under different physi- cochemical conditions. Limited proteolysis experiments coupled with mass spectrometric identification of cleavage fragments suggested a high degree of conformational flexibility affecting at least the first 21 residues of NS3. The accessibility of this region of the protease to limited chymotryptic digestion did not significantly change in any condition tested, whereas a significant reduction of chymotryptic cleavages within 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 with the cofactor peptide is highly flexible in solution and contributes only marginally to the thermodynamic stability of the complex. (2) Affinity enhancement is accomplished by several factors through a general stabilization of the fold of the NS3 molecule. Hepatitis C is a major health problem affecting about 0.5- 1.5% of the world's population. The disease is caused by a viral infectious agent, the hepatitis C virus (HCV 1 ), whose major route of transmission is parenteral (1-3). The virus was identified by molecular cloning in 1989 (1), and its genome, a 9.5-kilobase single-stranded positive sense RNA molecule, contains a single open reading frame encoding a polyprotein of 3010-3033 residues. Upon its synthesis in cells, this polyprotein is processed into the mature polypep- tides through the combined action of both cellular signal peptidases and two virally encoded proteolytic enzymes (reviewed in ref 4). The serine protease domain contained in the viral NS3 protein appears to play a prominent role in the processing of the HCV polyprotein. It is responsible for 4 out of the 5 cleavage events that have to take place in order to generate the mature nonstructural proteins believed to form the replication machinery of the virus. The NS3- dependent processing occurs at the intramolecular NS3/NS4A site and at the intermolecular NS4A/NS4B, NS4B/NS5A, and NS5A/NS5B cleavage sites of the HCV polyprotein. Besides containing a serine protease domain of about 20 kDa at its N-terminus, the NS3 protein harbors an RNA helicase in its 400 C-terminal residues (5). Deletion experiments have shown that helicase and protease domains can work independently of each other, with the separate polypeptide chains expressing the respective activities ( 5-12). This has recently permitted the crystallization of the NS3 protease and helicase domains (13-16).