title: Deciphering the molecular basis for nucleotide selection by the West Nile virus RNA helicase.
abstract: The West Nile virus RNA helicase uses the energy derived from the hydrolysis of nucleotides to separate complementary strands of RNA. Although this enzyme has a preference for ATP, the bias towards this purine nucleotide cannot be explained on the basis of specific protein-ATP interactions. Moreover, the enzyme does not harbor the characteristic Q-motif found in other helicases that regulates binding to ATP. In the present study, we used structural homology modeling to generate a model of the West Nile virus RNA helicase active site that provides instructive findings on the interaction between specific amino acids and the ATP substrate. In addition, we evaluated both the phosphohydrolysis and the inhibitory potential of a collection of 30 synthetic purine analogs. A structure-guided alanine scan of 16 different amino acids was also performed to clarify the contacts that are made between the enzyme and ATP. Our study provides a molecular rationale for the bias of the enzyme for ATP by highlighting the specific functional groups on ATP that are important for binding. Moreover, we identified three new essential amino acids (Arg-185, Arg-202 and Asn-417) that are critical for phosphohydrolysis. Finally, we provide evidence that a region located upstream of motif I, which we termed the nucleotide specificity region, plays a functional role in nucleotide selection which is reminiscent to the role exerted by the Q-motif found in other helicases.
authors.affiliation: RNA Group/Groupe ARN, Département de Biochimie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.
authors.forename: Simon
authors.initials: S
authors.lastname: Despins
authors.forename: Moheshwarnath
authors.initials: M
authors.lastname: Issur
authors.forename: Isabelle
authors.initials: I
authors.lastname: Bougie
authors.forename: Martin
authors.initials: M
authors.lastname: Bisaillon
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D020365:RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014902:West Nile virus / Q000201:enzymology*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D000255:Adenosine Triphosphate; D006160:Guanosine Triphosphate; D009711:Nucleotides; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases