RNA helicases of the DEAD (Asp-Glu-Ala-Asp)-box family of proteins are involved in many aspects of RNA metabolism from transcription to RNA decay, but most of them have also been shown to be multifunctional. The DEAD-box helicase DDX5 of host cells has been shown to interact with the RNA-dependent RNA polymerase (NS5B) of HCV (hepatitis C virus). In the present study, we report the presence of two independent NS5B-binding sites in DDX5, one located at the N-terminus and another at the C-terminus. The N-terminal fragment of DDX5, which consists of the first 305 amino acids and shall be referred as DDX5-N, was expressed and crystallized. The crystal structure shows that domain 1 (residues 79–303) of DDX5 contains the typical features found in the structures of other DEAD-box helicases. DDX5-N also contains the highly variable NTR (N-terminal region) of unknown function and the crystal structure reveals structural elements in part of the NTR, namely residues 52–78. This region forms an extensive loop and an α-helix. From co-immunoprecipitation experiments, the NTR of DDX5-N was observed to auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1–51 residues in NTR and the NS5B-binding site in DDX5-N. Furthermore, NMR investigations reveal that there is a direct interaction between DDX5 and NS5B in vitro.
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August 2012
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Research Article|
July 27 2012
The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus
Sujit Dutta;
Sujit Dutta
1
*Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673
†School of Life Sciences and Chemical Technology, Ngee Ann Polytechnic, Singapore 599489
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Garvita Gupta;
Garvita Gupta
1
‡Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543
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Yook-Wah Choi;
Yook-Wah Choi
*Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673
§Molecular Virology Laboratory, Department of Medical Biosciences, University of the Western Cape, Cape Town 7535, South Africa
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Masayo Kotaka;
Masayo Kotaka
∥Department of Physiology, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong
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Burtram C. Fielding;
Burtram C. Fielding
§Molecular Virology Laboratory, Department of Medical Biosciences, University of the Western Cape, Cape Town 7535, South Africa
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Jianxing Song;
Jianxing Song
‡Department of Biological Sciences, Faculty of Science, National University of Singapore, Singapore 117543
¶Department of Biochemistry, Yong Loo Lin School of Medicine, National University Health System (NUHS), National University of Singapore, Singapore 117597
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Yee-Joo Tan
Yee-Joo Tan
2
*Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), Singapore 138673
**Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597
2To whom correspondence should be addressed (email Yee_Joo_TAN@NUHS.edu.sg).
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Biochem J (2012) 446 (1): 37–46.
Article history
Received:
January 03 2012
Revision Received:
May 21 2012
Accepted:
May 28 2012
Accepted Manuscript online:
May 28 2012
Citation
Sujit Dutta, Garvita Gupta, Yook-Wah Choi, Masayo Kotaka, Burtram C. Fielding, Jianxing Song, Yee-Joo Tan; The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus. Biochem J 15 August 2012; 446 (1): 37–46. doi: https://doi.org/10.1042/BJ20120001
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