Design, high-level expression, purification and characterization of soluble fragments of the hepatitis C virus NS3 RNA helicase suitable for NMR-based drug discovery methods and mechanistic studies

RNA helicases represent a family of enzymes that unwind double-stranded(ds) RNA in a nucleoside triphosphate (NTP)-dependent fashionand which are required in all aspects of cellular RNA metabolismand processing. The hepatitis C virus (HCV) non-structural 3(NS3) protein possesses a serine protease activity in the N-terminalone-third, whereas RNA-stimulated NTPase and helicase activitiesreside in the C-terminal portion of the 631 amino acid residuebifunctional enzyme. The HCV NS3 RNA helicase is of key importancein the life cycle of HCV, which makes it a target for the developmentof therapeutics. However, neither the precise mechanism northe substrate structure has been defined for this enzyme. Fornuclear magnetic resonance (NMR)-based drug discovery methodsand for mechanistic studies we engineered, prepared and characterizedvarious truncated constructs of the 451-residue HCV NS3 RNAhelicase. Our goal was to produce smaller fragments of the enzyme,which would be amenable to solution NMR techniques while retainingtheir native NTP and/or nucleic acid binding sites. Solutionconditions were optimized to obtain high-quality heteronuclearNMR spectra of nitrogen-15 isotope-labeled constructs, whichare typical of well-folded monomeric proteins. Moreover, NMRbinding studies and functional data directly support the correctfolding of these fragments.