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 bet ween an 82-residue protein fragment containing the core conserved regi on of the glucocorticoid receptor DNA-binding domain (GR DBD) and a pa lindromic glucocorticoid response element (GRE), a consensus GRE half- site, a consensus estrogen response element (ERE) half-site, and two i ntermediate half-sites (GRE2 and ERE2). Equilibrium parameters were de termined at 20-degrees-C and buffer conditions that approximate intrac ellular conditions. The association constants for GR DBD binding to th e 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 concentratio ns of an antioxidant and a nonionic detergent are (1.0 +/- 0.1) X 10(6 ) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association con stants for binding to the ERE (5'TGACCT3') and ERE2 (5'TGATCT3') half- sites are < 10(5) M-1. The implications of these numbers for the speci ficity and affinity for the binding of the intact GR to DNA are discus sed. Comparison of GR DBD binding to a GRE half-site and a palindromic GRE sequence allowed us to estimate the cooperativity parameter, omeg a(obs) = 25-50, for GR DBD binding to GRE. The thermodynamics of the G R DBD interaction with a GRE half-site were also investigated by deter mining the temperature dependence of the observed association constant . The nonlinear dependence in ln K(obs), as a function of 1/T is consi stent with a change in standard heat capacity, DELTAC(p)degrees(obs) = 1.0 +/- 0.2 kcal mol-1 K-1. The binding process is shown to be entrop y driven at temperatures < 26-degrees-C and enthalpy driven at tempera tures > 35-degrees-C. The thermodynamics of the binding process are co nsistent with dehydration of nonpolar surfaces upon formation of the c omplex, although the observed DELTAC(p)degrees(obs) cannot be fully ac counted for by this mechanism.