title: Multiple determinants influence complex formation of the hepatitis C virus NS3 protease domain with its NS4A cofactor peptide.
abstract: 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 physicochemical 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.
authors.affiliation: Istituto di Ricerche di Biologia Molecolare, Rome, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Urbani
authors.forename: G
authors.initials: G
authors.lastname: Biasiol
authors.forename: M
authors.initials: M
authors.lastname: Brunetti
authors.forename: C
authors.initials: C
authors.lastname: Volpari
authors.forename: S
authors.initials: S
authors.lastname: Di Marco
authors.forename: M
authors.initials: M
authors.lastname: Sollazzo
authors.forename: S
authors.initials: S
authors.lastname: Orrú
authors.forename: F D
authors.initials: FD
authors.lastname: Piaz
authors.forename: A
authors.initials: A
authors.lastname: Casbarra
authors.forename: P
authors.initials: P
authors.lastname: Pucci
authors.forename: C
authors.initials: C
authors.lastname: Nardi
authors.forename: P
authors.initials: P
authors.lastname: Gallinari
authors.forename: R
authors.initials: R
authors.lastname: De Francesco
authors.forename: C
authors.initials: C
authors.lastname: Steinkühler
mesh_terms: D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003902:Detergents; D004795:Enzyme Stability; D005990:Glycerol / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D003902:Detergents; D005990:Glycerol; D046911:Macromolecular Substances; D010446:Peptide Fragments; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus