Site-Specific Cation Binding Mediates TATA Binding Protein-DNA Interaction from a Hyperthermophilic Archaeon
详细信息 查看全文
- 作者：Simon Bergqvist ; Ronan O'Brien ; and John E. Ladbury
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：February 27, 2001
- 年：2001
- 卷：40
- 期：8
- 页码：2419 - 2425
- 全文大小：465K
- 年卷期：v.40,no.8(February 27, 2001)
- ISSN：1520-4995
文摘
Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditionsof high salt and elevated temperature. In a previous study [O'Brien, R., DeDecker, B., Fleming, K., Sigler,P. B., and Ladbury, J. E., (1998) J. Mol. Biol. 279, 117-125], we showed that, despite the similarity ofprimary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamicallyin a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increasesas the salt concentration is increased. The formation of the protein-DNA complex involves the releaseof water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis byselecting potential cation binding sites at negatively charged, acidic residues in the complex interface.These were substituted using site-directed mutagenesis of specific residues. Changes in the thermodynamicparameters on formation of the mutant protein-DNA complex were determined using isothermal titrationcalorimetry and compared to the wild type interaction. Removal of a glutamate residue from the bindingsite resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directlyinvolved in the binding of cations in the complex interface. Substitution of another acidic residue proximalto the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the aminoacid on the protein surface is important in dictating the potential to coordinate cations. Removal of thecation binding site provided a more favorable entropy of binding; however, this effect is significantlyreduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinitywith respect to the wild-type TBP at low salt concentrations.