title: Hepatitis C virus NS3 ATPase/helicase: an ATP switch regulates the cooperativity among the different substrate binding sites.
abstract: The protease/helicase NS3 is believed to play a central role in the replication cycle of the hepatitis C virus (HCV), and, therefore, it is an attractive target for antiviral chemotherapy. Several enzymological studies and crystallographic structures are available for the NS3 protease and helicase domains individually, but less is known about the NTPase and helicase activities of the full-length protein. The aim of our study was to characterize from an enzymological point of view the mechanism of interaction of the full-length NS3 protease/helicase with its nucleic acid (NA) and ATP substrates. Our kinetic analysis revealed that both the NA and ATP substrates can interact cooperatively with the enzyme through the coordinated action of two binding sites. Moreover, the observation of a reciprocal influence of both substrates on the kinetics of their interaction with the enzyme suggested that the NS3 helicase works as a dimer which can exist in three functionally different states: (i) an unbound state, with two equivalent low-affinity binding sites for ATP, which shows cooperative high-affinity NA binding; (ii) an ATP-bound state, with two equivalent low-affinity NA binding sites; and (iii) a NA-bound state, with two equivalent high-affinity ATP binding sites. The cycling between these different conformational states is thus regulated by an ATP switch. These results are discussed in light of the current models for NA unwinding by the HCV NS3 helicase.
authors.affiliation: Istituto di Genetica Molecolare, IGM-CNR, I-27100 Pavia, Italy.
authors.forename: Giada Aurora
authors.initials: GA
authors.lastname: Locatelli
authors.forename: Silvio
authors.initials: S
authors.lastname: Spadari
authors.forename: Giovanni
authors.initials: G
authors.lastname: Maga
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D004277:DNA, Single-Stranded / Q000737:chemistry; D019281:Dimerization; D016174:Hepacivirus / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009841:Oligonucleotides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry*; D012697:Serine Endopeptidases / Q000737:chemistry; D013053:Spectrophotometry; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D009841:Oligonucleotides; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases