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Crystal structure of TBP-interacting protein (Tk-TIP26) and implications for its inhibition mechanism of the interaction between TBP and TATA-DNA

¹ Department of Applied Chemistry and ² Department of Material and Life Science, Graduate School of Engineering, Osaka University, Suita, Osaka 565-0871, Japan

(RECEIVED August 17, 2005; FINAL REVISION October 14, 2005; ACCEPTED October 14, 2005)

TATA-binding protein (TBP)-interacting protein from the hyperthermophilic archaeon Thermococcus kodakaraensis strain KOD1 (Tk-TIP26) is a possible transcription regulatory protein in Thermococcales. Here, we report the crystal structure of Tk-TIP26 determined at 2.3 Å resolution with multiple-wavelength anomalous dispersion (MAD) method. The overall structure of Tk-TIP26 consists of two domains. The N-terminal domain forms an / structure, in which three -helices enclose the central -sheet. The topology of this domain is similar to that of holliday junction resolvase Hjc from Pyrococcus furiosus. TheC-terminal domain comprises three -helices, six -strands, and two 3₁₀-helices. In the dimer structure of Tk-TIP26, two molecules are related with the crystallographic twofold axis, and these molecules rigidly interact with each other via hydrogen bonds. The complex of Tk-TIP26/Tk-TBP is isolated and analyzed by SDS-PAGE and gel filtration column chromatography, resulting in a stoichiometric ratio of the interaction between Tk-TIP26 and Tk-TBP of 4:2, i.e., two dimer molecules of Tk-TIP26 formed a complex with one dimeric TBP. The electrostatic surfaces of Tk-TIP26 and TBP from Pyrocuccus woesei (PwTBP) allowed us to build a model of the Tk-TIP26/TBP complex, and to propose the inhibition mechanism where two dimer molecules of Tk-TIP26 bind to a dimeric TBP, preventing its binding to TATA-DNA.

Keywords: transcription; TATA-binding protein (TBP); TBP-interacting protein; hyperthermophilic archaeon; X-ray crystallography; Cys₂–His₂ type zinc finger motif; gel filtration chromatography; protein structure/folding; structure/function studies; protein crystallization; docking proteins

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051788906.

Reprint requests to: Yasushi Kai, Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; e-mail: kai{at}chem.eng.osaka-u.ac.jp; fax: +81-6-6879-7409.

³ Present addresses: Laboratory for Nanosystems Physiology, Research Institute for Electronic Science, Hokkaido University, Sapporo, 060-0812, Japan;

⁴ Laboratory of Environmental Molecular Biology, Graduate School of Environmental Earth Science, Hokkaido University, Sapporo 060-0810, Japan.

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