Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon.

Pyrococcus woesei (Pw) is a hyperthermophilic archaeal organism that exists under conditions of 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 of primary and secondary structure, the TATA box binding protein (TBP) from Pw binds thermodynamically in a fundamentally different way to its mesophilic counterparts. The affinity of the interaction increases as the salt concentration is increased. The formation of the protein-DNA complex involves the release of water and the uptake of ions, which were hypothesized to be cations. Here we test this hypothesis by selecting 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 thermodynamic parameters on formation of the mutant protein-DNA complex were determined using isothermal titration calorimetry and compared to the wild type interaction. Removal of a glutamate residue from the binding site resulted in the uptake of one less cation on formation of the complex. This glutamate (E12) is directly involved in the binding of cations in the complex interface. Substitution of another acidic residue proximal to the DNA binding site (D101) had no effect on cation uptake, suggesting that the location of the amino acid on the protein surface is important in dictating the potential to coordinate cations. Removal of the cation binding site provided a more favorable entropy of binding; however, this effect is significantly reduced at higher salt concentrations. The removal of the cation binding site led to an increase in affinity with respect to the wild-type TBP at low salt concentrations.