Towards the design of flavivirus helicase/NTPase inhibitors: crystallographic and mutagenesis studies of the dengue virus NS3 helicase catalytic domain

Novartis Found Symp. 2006:277:87-97; discussion 97-101, 251-3. doi: 10.1002/0470058005.ch7.

Abstract

Infectious diseases caused by flaviviruses are important emerging public health concerns and new vaccines and therapeutics are urgently needed. The NS3 protein from flavivirus is a multifunctional protein with protease, helicase and nucleoside 5' triphosphatase activities (NTPase). Thus, NS3 plays a crucial role in viral replication and represents an interesting target for the development of specific antiviral inhibitors. We have solved the structure of an enzymatically active fragment of the dengue virus NTPase/ helicase C-terminal catalytic domain in several related crystal forms. The structure is composed of three domains, bears an asymmetric distribution of charges and comprises a tunnel large enough to accommodate single strand RNA. A concave face formed by domains 2 and 3 is proposed to bind a nucleic acid duplex substrate. Comparison of the various copies of dengue and yellow fever virus NS3 NTPase/helicase catalytic domains reveals mobile regions of the enzyme. Such dynamic behaviour is likely to be coupled with directional translocation along the single strand nucleic acid substrate during strand separation. We used structure-based site directed mutagenesis to identify regions of the enzyme that are crucial for its ATPase or nucleic acid duplex unwinding activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Catalytic Domain
  • Crystallization
  • DNA Helicases / antagonists & inhibitors
  • DNA Helicases / chemistry*
  • DNA Helicases / genetics
  • Dengue Virus / enzymology*
  • Dimerization
  • Drug Design*
  • Enzyme Inhibitors / chemistry*
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Nucleoside-Triphosphatase / antagonists & inhibitors
  • Nucleoside-Triphosphatase / chemistry*
  • Nucleoside-Triphosphatase / genetics
  • RNA Helicases / chemistry
  • RNA Helicases / genetics
  • RNA Helicases / metabolism
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / metabolism
  • Viral Nonstructural Proteins / chemistry*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism

Substances

  • Enzyme Inhibitors
  • NS3 protein, flavivirus
  • Viral Nonstructural Proteins
  • Serine Endopeptidases
  • Nucleoside-Triphosphatase
  • DNA Helicases
  • RNA Helicases