Browsing by Author "Dutta, Sujit"

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    Expression, purification and preliminary crystallographic analysis of recombinant human DEAD-box polypeptide 5
    (International Union of Crystallography, 2010) Choi, Yook-Wah; Dutta, Sujit; Fielding, Burtram C.; Tan, Yee-Joo
    The DEAD-box RNA helicase DDX5 is involved in many aspects of RNA processing and has been implicated in a number of cellular processes involving alteration of RNA secondary structure. The N-terminal region of DDX5, which contains the conserved domain 1 of the DEAD-box helicases, has been cloned and expressed in Escherichia coli and purified. Here, the crystallization and preliminary diffraction analysis of this region is reported. X-ray diffraction data were processed to a resolution of 2.7 A ° . The crystals belonged to space group I222, with unit-cell parameters a = 66.18, b = 73.80, c = 104.00 A ° , = = = 90 .
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    The variable N-terminal region of DDX5 contains structural elements and auto-inhibits its interaction with NS5B of hepatitis C virus
    (Portland Press, 2012) Dutta, Sujit; Gupta, Garvita; Choi, Yook-Wah; Kotaka, Masayo; Fielding, Burtram C.; Song, Jianxing; Tan, Yee-Joo
    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 NS5Bbinding 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 DEADbox 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.