The metal binding site of the hepatitis C virus NS3 protease: A spectroscopic investigation

Andrea Urbani, Renzo Bazzo, Maria Chiara Nardi, Daniel Oscar Cicero, Raffaele De Francesco, Christian Steinkühler, Gaetano Barbato

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Abstract

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 δ-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.

Original languageEnglish
Pages (from-to)18760-18769
Number of pages10
JournalJournal of Biological Chemistry
Volume273
Issue number30
DOIs
Publication statusPublished - Jul 24 1998

ASJC Scopus subject areas

  • Biochemistry

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    Urbani, A., Bazzo, R., Nardi, M. C., Cicero, D. O., De Francesco, R., Steinkühler, C., & Barbato, G. (1998). The metal binding site of the hepatitis C virus NS3 protease: A spectroscopic investigation. Journal of Biological Chemistry, 273(30), 18760-18769. https://doi.org/10.1074/jbc.273.30.18760