COMPLEX-FORMATION BETWEEN THE HEPATITIS-C VIRUS SERINE-PROTEASE AND ASYNTHETIC NS4A COFACTOR PEPTIDE

The NS3 protein of the hepatitis C virus contains a serine protease th at, upon binding to its cofactor, NS4A, is responsible for maturationa l cleavages that occur in the nonstructural region of the viral polypr otein. We have studied in vitro complex formation between the NS3 prot ease domain expressed in Escherichia coli and a synthetic peptide span ning the minimal domain of the NS4A cofactor. Complex dissociation con stants in the low micromolar range were measured using different techn iques such as activity titration, fluorescence titration, and pre-equi librium analysis of complex formation. Cofactor binding was strictly d ependent on the glycerol content of buffer solutions and was not signi ficantly influenced by substrate saturation of the enzyme. NS4A peptid e binding to NS3 was accompanied by changes in the circular dichroism spectrum in the region between 270 and 290 nm, as well as by an enhanc ement of tryptophan fluorescence. Conversely, no changes in the far UV region of the circular dichroism spectrum were detectable. These data are indicative of induced tertiary structure changes and suggest that the secondary structure content of the uncomplexed enzyme does not di ffer significantly from that of the NS3-cofactor complex. Pre-equilibr ium measurements of complex formation showed very low values for k(on) , suggesting conformational transitions to be rate limiting for the as sociation reaction.