Flaviviral Helicases: Structure, Function, Inhibition and Dynamics

Abstract

Flaviviruses are ssRNA viruses causing several human diseases such as Dengue and Yellow fever. Helicases (Hel) are motor proteins involved in nucleotide remodeling. During viral replication the new formed dsRNA (template+daughter) is unwound by Hel. A molecular picture describing how the chemical energy derived from ATP hydrolysis is transformed into mechanical actions required for dsRNA unwinding is still missing.Flaviviral Hel is a monomeric protein composed of three domains hosting an internal cleft that bind ssRNA. From the crystal structure of Kunjin virus helicase (K-Hel; Mastrangelo et al., 2007) we hypothesized that the ssRNA entrance site was located between domains II and III. We evaluated such site as an attractive target for protein inhibition. Using in silico docking we identify a new compound, proved to be active against flaviviruses in cell culture (patent EP09174368).Normal mode analysis of K-Hel suggested a ‘scissor-like’ domain rearrangement likely involved in protein activity. Accordingly, the structures of Kokobera Hel in two crystal forms showed open and closed conformations of the ssRNA access site (unpublished results).In order to investigate such dynamical features we performed two molecular dynamics simulations of Dengue Hel in the presence and absence of ATP. The dynamics showed that ATP induces the closure of the ssRNA access site. The mechanical feature of the signal transmission between ATP binding site and ssRNA access site, located about 30 A apart, are described and a novel role of Hel motif V is underlined. Preliminary SAXS experiment confirmed the role of ATP in inducing protein closure, showing a slightly reduced radius of gyration. Protein mutants will be produced to analyze the ‘transmission shaft’ from ATP binding site to ssRNA access site.