Affinity labeling of hepatitis C virus replicase with a nucleotide analogue: identification of binding site.

We have used an ATP analogue 5′-[p-(fluorosulfonyl)benzoyl]adenosine (FSBA) to modify HCV replicase in order to identify the ATP binding site in the enzyme. FSBA inactivates HCV replicase activity in a concentration-dependent manner with a binding stoichiometry of 2 moles of FSBA per mole of enzyme. The enzyme activity is protected from FSBA in the presence of rNTP substrates or double-stranded RNA template primers that do not support ATP as the incoming nucleotide but not in the presence of polyrU.rA26. HPLC analysis of tryptic peptides of FSBA-modified enzyme revealed the presence of two distinct peptides eluted at 23 and 36 min; these were absent in the control. Further we noted that both peptides were protected from FSBA modification in the presence of Mg·ATP. The LC/MS/MS analysis of the affinity-labeled tryptic peptides purified from HPLC, identified two major modification sites at positions 382 (Tyr), and 491 (Lys) and a minor site at position 38 (Tyr). To validate the functional significance of Ty...