Thermodynamic analysis of the interaction between dengue virus NS3 helicase and ssRNA

LEOPOLDO G GEBHARD; MARIANA GALLO; J JEREMÍAS INCICCO; ANDREA V GAMARNIK; SERGIO B KAUFMAN

Cambridge

Conferencia; EMBO Conference - Helicases and Nucleic Acid Translocases Co-sponsored by the Harden Conferences.; 2013

EMBO

two functional domains: an N-terminal region (residues 1?167) that forms a two-component serine protease domain and a C-terminal region (residues 171?618) endowed with RNA-helicase, nucleoside 5′-triphosphatase (NTPase) and 5′-terminal RNA triphosphatase (RTPase) activities [1- 3]. These two functional domains may act independently of each other in vitro and recombinant NS3h lacking the protease domain retains NTPase and helicase activities [4]. The structure of DENV NS3h is characterized by three domains. The first two (I and II), are RecAlike domains, which constitute the core of most ATP-driven molecular motors [5], and host seven characteristic sequence motifs of superfamily 2 DExH helicases. The cleft formed between domain III and the other two presents numerous basic residues and is wide enough to accommodate a single-stranded nucleic acid substrate but not a duplex [6]. RNA helicases are ubiquitous enzymes that participate in virtually all processes of RNA metabolism. A common feature shared by these motor proteins is their capability to catalyze the hydrolysis of nucleoside triphosphates (NTPs) [7], which provides the driving force for the rearrangement of the RNA structures. In this poster we report results from Pulse field gradient NMR (PFG-NMR) methods to determine the oligomeric state of NS3h in solution, and quantitative fluorescence titration experiments performed to study the interaction of NS3h with ssRNA.