Multiple Determinants Influence Complex Formation of the Hepatitis C Virus NS3 Protease Domain with Its NS4A Cofactor Peptide

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• 作者：Andrea Urbani ; Gabriella Biasiol ; Mirko Brunetti ; Cinzia Volpari ; Stefania Di Marco ; Maurizio Sollazzo ; Stefania Orrú ; Fabrizio Dal Piaz ; Annarita Casbarra ; Piero Pucci ; Chiara Nardi ; Paola Gal
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：April 20, 1999
• 年：1999
• 卷：38
• 期：16
• 页码：5206 - 5215
• 全文大小：147K
• 年卷期：v.38,no.16(April 20, 1999)
• ISSN：1520-4995

文摘

The interaction of the hepatitis C virus (HCV) NS3 protease domain with its NS4A cofactorpeptide (Pep4AK) was investigated at equilibrium and at pre-steady state under different physicochemicalconditions. Equilibrium dissociation constants of the NS3-Pep4AK complex varied by several orders ofmagnitude depending on buffer additives. Glycerol, NaCl, detergents, and peptide substrates were foundto stabilize this interaction. The extent of glycerol-induced stabilization varied in an HCV strain-dependentway with at least one determinant mapping to an NS3-NS4A interaction site. Conformational transitionsaffecting at least the first 18 amino acids of NS3 were the main energy barriers for both the associationand the dissociation reactions of the complex. However, deletion of this N-terminal portion of the proteasemolecule only slightly influenced equilibrium dissociation constants determined under different physicochemical conditions. Limited proteolysis experiments coupled with mass spectrometric identificationof cleavage fragments suggested a high degree of conformational flexibility affecting at least the first 21residues of NS3. The accessibility of this region of the protease to limited chymotryptic digestion did notsignificantly change in any condition tested, whereas a significant reduction of chymotryptic cleavageswithin the NS3 core was detected under conditions of high NS3-Pep4AK complex affinity. We concludethe 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 onlymarginally to the thermodynamic stability of the complex. (2) Affinity enhancement is accomplished byseveral factors through a general stabilization of the fold of the NS3 molecule.