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 fashion and which are required in all aspects of cellular RNA metabolism and processing. The hepatitis C virus (HCV) non-structural 3 (NS3) protein possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion of the 631 amino acid residue bifunctional enzyme. The HCV NS3 RNA helicase is of key importance in the life cycle of HCV, which makes it a target for the development of therapeutics. However, neither the precise mechanism nor the substrate structure has been defined for this enzyme. For nuclear magnetic resonance (NMR)-based drug discovery methods and for mechanistic studies we engineered, prepared and characterized various truncated constructs of the 451-residue HCV NS3 RNA helicase. Our goal was to produce smaller fragments of the enzyme, which would be amenable to solution NMR techniques while retaining their native NTP and/or nucleic acid binding sites. Solution conditions were optimized to obtain high-quality heteronuclear NMR spectra of nitrogen-15 isotope-labeled constructs, which are typical of well-folded monomeric proteins. Moreover, NMR binding studies and functional data directly support the correct folding of these fragments.
Gesell
J J
JJ
These two authors contributed equally to this work. Department of Structural Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.
Liu
D
D
Madison
V S
VS
Hesson
T
T
Wang
Y S
YS
Weber
P C
PC
Wyss
D F
DF
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D000998:Antiviral Agents / Q000737:chemistry; D015195:Drug Design; D007700:Kinetics; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012995:Solubility; D014161:Transduction, Genetic; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000998:Antiviral Agents; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases