title: Characterisation of the role of zinc in the hepatitis C virus NS2/3 auto-cleavage and NS3 protease activities.
abstract: Cleavage of the hepatitis C virus polyprotein between the non-structural NS2 and NS3 proteins is mediated by a poorly characterised auto-proteolytic activity that maps to the C terminus of NS2 and the N terminus of NS3, but is distinct from the NS3 protease activity responsible for downstream cleavages in the polyprotein. We have exploited the fact that the minimal precursor (residues 904-1206 of the HCV polyprotein) can be expressed as an insoluble protein in Escherichia coli and subsequently refolded into a form active for both auto-cleavage and NS3 protease activity, to further characterise the NS2/3 auto-cleavage activity. We show that both activities are zinc-dependent and show an absolute requirement for cysteine residues 1123, 1125 and 1171 within NS3. In contrast cysteine 922 (within NS2) is only required for NS2/3 auto-cleavage activity and histidine 1175 is only required for NS3 activity. Although the complete NS3 protease domain (including the C-terminal alpha-helix) is required for NS2/3 auto-cleavage, the activity of the NS3 protease is not essential. Lastly we show that the NS2/3 auto-cleavage activity is more sensitive to zinc chelation by 1,10-phenanthroline than the NS3 protease activity. This observation is consistent with different conformations of the precursor competent for either NS2/3 auto-cleavage or NS3 protease activity; these two conformations can be distinguished by their relative strength and geometry of zinc coordination.
authors.affiliation: Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Philip R
authors.initials: PR
authors.lastname: Tedbury
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
mesh_terms: D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D016174:Hepacivirus / Q000201:enzymology* / Q000378:metabolism; D011489:Protein Denaturation; D011498:Protein Precursors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D011498:Protein Precursors; D017361:Viral Nonstructural Proteins; D015032:Zinc; D003545:Cysteine; D000409:Alanine