THE METAL-BINDING SITE OF THE HEPATITIS-C VIRUS NS3 PROTEASE - A SPECTROSCOPIC INVESTIGATION

The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural r egion of the viral polyprotein, The minimal domain with proteolytic ac tivity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray c rystallography as being three cysteines (Cys(97), Cys(99), and Cys(145 )) and one histidine residue (His(149)), which is postulated to coordi nate the metal through a water molecule. In this article, we present a n analysis of the role of metal coordination with respect to enzyme ac tivity and folding. Using NMR spectroscopy, the resonances of His149 w ere assigned based on their isotropic shift in a Co(II)-substituted pr otein. Data obtained with N-15-labeled NS3 protease were compatible wi th the involvement of the delta-N of His(149) in metal coordination. p H titration experiments showed that the cooperative association of at least two protons is required in the protonation process of His149. Ch anges in the NMR signals of this residue between pH 7 and 5 are interp reted as evidence for a structural change at the metal binding site, w hich switches from a ''closed'' to an ''open'' conformation. Site-dire cted mutagenesis of His149 has shown the importance of this residue in the metal incorporation pathway and for achieving an active fold. The metal coordination of the protease was also investigated by circular dichroism and electronic absorption spectroscopies using a Co(II)-subs tituted enzyme. We show evidence for rearrangements of the metal coord ination geometry induced by complex formation with an NS4A peptide cof actor. No such changes were observed upon binding to a substrate pepti de. Also, CN- and N-3(-) induced Co(II) ligand field perturbations, wh ich went along with an 1.5-fold enhancement of protease activity.