Characterization of the metal ion binding properties of the hepatitis C virus RNA polymerase.
The hepatitis C virus nonstructural 5B protein (NS5B) protein has been shown to require either magnesium or manganese for its RNA-dependent RNA polymerase activity. As a first step toward elucidating the nature and the role(s) of the metal ions in the reaction chemistry, we have utilized endogenous tryptophan fluorescence to quantitate the interactions of magnesium and manganese ions with this protein. The association of either Mg(2+) or Mn(2+) ions with the enzyme resulted in a decrease in the intensity of the tryptophan emission spectrum. This decrease was used to determine the apparent dissociation constants for both ions. The apparent K(d) values for the binding of Mg(2+) and Mn(2+) ions to the free enzyme were 3.1 and 0.3 mm, respectively. Dual ligand titration experiments demonstrated that both ions bind to a single common site, for which they compete. The kinetics of real time metal ion binding to the NS5B protein were also investigated. Based on the results of our fluorescence and near-UV circular dichroism experiments, we show that NS5B undergoes conformational changes upon the binding of metal ions. However, this process does not significantly stimulate the binding to the RNA or NTP substrates. We envisage that the ion-induced conformational change is a prerequisite for catalytic activity by both correctly positioning the side chains of the residues located in the active site of the enzyme and also contributing to the stabilization of the intermediate transition state.
Bougie
Isabelle
I
Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Québec J1H 5N4, Canada.
Charpentier
Sébastien
S
Bisaillon
Martin
M
D002413:Cations, Divalent; D002942:Circular Dichroism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D016174:Hepacivirus / Q000201:enzymology*; D007700:Kinetics; D008670:Metals / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase
D002413:Cations, Divalent; D012321:DNA-Directed RNA Polymerases; D008670:Metals; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase