Insights into RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein

Abstract

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 catalyticcompetent 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 b-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.

Flaviviral helicase: Insights into the mechanism of action of a motor protein

“…Since the 3 10 helix of motif V (induced by ATP binding) is in contact with ssRNA along its phosphate chain [4], we can speculate that the horizontal oscillations observed for motif V in presence/ absence of ATP (Fig. 2) might be one of the main components of the driving force pulling the protein along ssRNA.…”

“…The available experimental evidences based on protein crystal structures [4] show only two different structural states of F-Hel that appear not to depend on the ligation state of the ATP binding site, but only on the presence/absence of ssRNA. In fact, if we take as reference the apo structure (PDB ID: 2JLQ) the r.m.s.…”

“…The overall F-Hel structure resembles that of an equilateral triangle, where each domain is placed at a vertex. The center of the triangle, where the three domains converge, contains an evident cleft that was first hypothesized [3], and then observed, to host ssRNA during protein activity [4]. The ssRNA access site in the cleft is located between a-helix 2 of domain II, the b-hairpin protruding from domain II toward domain III, and the C-terminal a-helix 6 of domain III; being surrounded by two a-helices, we will refer to the ssRNA access site as the ''a-helical gate''.…”

“…The molecular dynamics (MD) simulations started from the atomic coordinates of F-Hel from Dengue virus serotype 4 (451 amino acids; PDB ID: 2JLQ, [4]). Using the program psfgen (part of the namd2 package, [11]) H-atoms were added and partial charges assigned to every atom in the structure.…”

“…High resolution results have been reported for Dengue virus Hel (DV-Hel) [4] and Hepatitis C virus Hel (HCV-Hel [6][7][8]). In particular, the recent publication on HCV-Hel [8] has unraveled the protein structural changes induced by ATP analogs mimicking the transition state occurring during ATP hydrolysis.…”