Sequence-Specific DNA Binding by the Glucocorticoid Receptor DNA-Binding Domain Is Linked to a Salt-Dependent Histidine Protonation

摘要

We used isothermal titration calorimetry in the temperature range 21-25degC to investigate the effect of pH on the calorimetric enthalpy (triangle openH_(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of triangle openH_(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK_a for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where triangle openH_(cal) shows a characteristic dependence on the solution pH. triangle openH_(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar triangel openH_(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in triangle openH_(cal) can be quantitatively described by a shift of the pK_a of His451 from appoximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na~+ counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.