Thermodynamics of the Glucocorticoid Receptor—DNA Interaction: Binding of Wild-Type GR DBD to Different Response Elements

We used fluorescence spectroscopy to study the chemical equilibria between an 82-residue protein fragment containing the core conserved region of the glucocorticoid receptor DNA-binding domain (GRDBD) and a palindromic glucocorticoid response element (GRE), a consensus GRE half-site, a consensus estrogen response element (ERE) half-site, and two intermediate half-sites (GRE2 and ERE2). Equilibrium parameters were determined at 20°C and buffer conditions that approximate intracellular conditions. The association constants for GR DBD binding to the GRE (5′TGTTCT3′) and GRE2 (5′TGTCCT3′) half-sites at 85 mM NaCl, 100 mM KCl, 2 mM MgCl₂, and 20 mM Tris-HCl at pH 7.4 and low concentrations of an antioxidant and a nonionic detergent are (1.0 ± 0.1) × 10⁶M^-1 and (5.1 ± 0.2) × 10⁵M^-1, respectively. The association constants for binding to the ERE (5′TGACCT3′) and ERE2 (5′TGATCT3′) half-sites are <10⁵ M^-1. The implications of these numbers for the specificity and affinity for the binding of the intact GR to DNA are discussed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, ω_obs = 25-50, for GR DBD binding to GRE. The thermodynamics of the GR DBD interaction with a GRE half-site were also investigated by determining the temperature dependence of the observed association constant. The nonlinear dependence in ln K_obs as a function of 1/T is consistent with a change in standard heat capacity, ΔC_p°_obs = 1.0 ± 0.2 kcal mol^-1 K^-1. The binding process is shown to be entropy driven at temperatures <26°C and enthalpy driven at temperatures >35°C. The thermodynamics of the binding process are consistent with dehydration of nonpolar surfaces upon formation of the complex, although the observed ΔC_p°_obs cannot be fully accounted for by this mechanism.