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

Thomas Lundbäck, Carol Cairns, Torleif Härd, Jan Ake Gustafsson, Jan Carlstedt-Duke

Research output: Contribution to journalArticle

50 Scopus citations

Abstract

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, ωobs = 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, ΔCp°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 ΔCp°obs cannot be fully accounted for by this mechanism.

Original languageEnglish (US)
Pages (from-to)5074-5082
Number of pages9
JournalBiochemistry
Volume32
Issue number19
DOIs
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry

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