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

Abstract

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 α/β structure, in which three α‐helices enclose the central β‐sheet. The topology of this domain is similar to that of holliday junction resolvase Hjc from Pyrococcus furiosus . The C‐terminal domain comprises three α‐helices, six β‐strands, and two 3 10 ‐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 ( Pw TBP) 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.