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 (GR DBD) 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 degrees 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) x 10(6) M-1 and (5.1 +/- 0.2) x 10(5) M-1, respectively. The association constants 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 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, omega 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 Kobs as a function of 1/T is consistent with a change in standard heat capacity, delta Cp degree obs = 1.0 +/- 0.2 kcal mol-1 K-1.(ABSTRACT TRUNCATED AT 250 WORDS)
Lundbäck
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Center for Structural Biochemistry, Karolinska Institutet, NOVUM, Huddinge, Sweden.
Cairns
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C
Gustafsson
J A
JA
Carlstedt-Duke
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Härd
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T
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004967:Estrogens / Q000494:pharmacology; D005938:Glucocorticoids / Q000494:pharmacology; D008969:Molecular Sequence Data; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D004967:Estrogens; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D011994:Recombinant Proteins; D004247:DNA