Towards the design of flavivirus helicase/NTPase inhibitors: crystallographic and mutagenesis studies of the dengue virus NS3 helicase catalytic domain.
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.
Xu
Ting
T
School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive Singapore 637551.
Sampath
Aruna
A
Chao
Alex
A
Wen
Daying
D
Nanao
Max
M
Luo
Dahai
D
Chene
Patrick
P
Vasudevan
Subhash G
SG
Lescar
Julien
J
D001665:Binding Sites; D020134:Catalytic Domain; D003460:Crystallization; D004265:DNA Helicases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D003716:Dengue Virus / Q000201:enzymology*; D019281:Dimerization; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D043583:Nucleoside-Triphosphatase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D004265:DNA Helicases; D004791:Enzyme Inhibitors; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases