Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein.
Together with the NS5 polymerase, the NS3 helicase has a pivotal function in flavivirus RNA replication and constitutes an important drug target. We captured the dengue virus NS3 helicase at several stages along the catalytic pathway including bound to single-stranded (ss) RNA, to an ATP analogue, to a transition-state analogue and to ATP hydrolysis products. RNA recognition appears largely sequence independent in a way remarkably similar to eukaryotic DEAD box proteins Vasa and eIF4AIII. On ssRNA binding, the NS3 enzyme switches to a catalytic-competent state imparted by an inward movement of the P-loop, interdomain closure and a change in the divalent metal coordination shell, providing a structural basis for RNA-stimulated ATP hydrolysis. These structures demonstrate for the first time large quaternary changes in the flaviviridae helicase, identify the catalytic water molecule and point to a beta-hairpin that protrudes from subdomain 2, as a critical element for dsRNA unwinding. They also suggest how NS3 could exert an effect as an RNA-anchoring device and thus participate both in flavivirus RNA replication and assembly.
Luo
Dahai
D
Structural & Computational Biology Division, School of Biological Sciences, Nanyang Technological University, Singapore.
Xu
Ting
T
Watson
Randall P
RP
Scherer-Becker
Daniella
D
Sampath
Aruna
A
Jahnke
Wolfgang
W
Yeong
Sui Sum
SS
Wang
Chern Hoe
CH
Lim
Siew Pheng
SP
Strongin
Alex
A
Vasudevan
Subhash G
SG
Lescar
Julien
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D012313:RNA / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry* / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000255:Adenosine Triphosphate; D012313:RNA; 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