The conformational stability of the rat thyroid transcription factor 1 homeodomain, TTF-1HD, has been investigated by means of circular dichroism (CD) and differential scanning calorimetry (DSC) measurements at pH 5.0 as a function of KCl concentration. Thermal unfolding of TTF-1HD is a reversible two-state transition. The protein is not stable against temperature, showing a denaturation temperature of 32 degrees C in the absence of salt and 50 degrees C at 75 mM KCl. The binding energetics of TTF-1HD to its target DNA sequence has been characterized by means of isothermal titration calorimetry (ITC) measurements, complemented with CD data. At 25 degrees C, pH 5.0 and 75 mM KCl, the binding constant amounts to 1.5 x 10(8)M(-1) and the binding enthalpy change amounts to -41 kJ mol(-1). The process is enthalpy driven, but also the entropy change is favorable to complex formation. To gain a molecular level understanding of the interactions determining the association of TTF-1HD to the target DNA sequence structural information would be requested, but it is not yet available. Therefore, structural models of two complexes, TTF-1HD with the target DNA sequence and TTF-1HD with a modified DNA sequence, have been constructed by using as a template the NMR structure of the complex between NK-2 HD and its target DNA, and by performing molecular dynamics simulations 3.5 ns long. Analysis of these models allows one to shed light on the origin of the DNA binding specificity characteristic of TTF-1HD.
(c) 2007 Wiley-Liss, Inc.