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J Biol Chem, Vol. 273, Issue 30, 18760-18769, July 24, 1998

The Metal Binding Site of the Hepatitis C Virus NS3 Protease
A SPECTROSCOPIC INVESTIGATION

From the Istituto di Ricerche di Biologia Molecolare "P. Angeletti," Via Pontina Km 30.600, 00040 Pomezia, Rome, Italy

The NS3 region of the hepatitis C virus encodes for a serine protease activity, which is necessary for the processing of the nonstructural region of the viral polyprotein. The minimal domain with proteolytic activity resides in the N terminus, where a structural tetradentate zinc binding site is located. The ligands being been identified by x-ray crystallography as being three cysteines (Cys⁹⁷, Cys⁹⁹, and Cys¹⁴⁵) and one histidine residue (His¹⁴⁹), which is postulated to coordinate the metal through a water molecule. In this article, we present an analysis of the role of metal coordination with respect to enzyme activity and folding. Using NMR spectroscopy, the resonances of His¹⁴⁹ were assigned based on their isotropic shift in a Co(II)-substituted protein. Data obtained with ¹⁵N-labeled NS3 protease were compatible with the involvement of the -N of His¹⁴⁹ in metal coordination. pH titration experiments showed that the cooperative association of at least two protons is required in the protonation process of His¹⁴⁹. Changes in the NMR signals of this residue between pH 7 and 5 are interpreted as evidence for a structural change at the metal binding site, which switches from a "closed" to an "open" conformation. Site-directed 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)-substituted enzyme. We show evidence for rearrangements of the metal coordination geometry induced by complex formation with an NS4A peptide cofactor. No such changes were observed upon binding to a substrate peptide. Also, CN and N₃ induced Co(II) ligand field perturbations, which went along with an 1.5-fold enhancement of protease activity.

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