Thermodynamics of Sequence-Specific Glucocorticoid Receptor-DNA Interactions

Thomas Lundb¤ck, Torleif Hard, Johanna Zilliacus, Jan Åke Gustafsson, Jan Carlstedt-Duke

Research output: Contribution to journalArticle

38 Scopus citations

Abstract

The thermodynamics of sequence-specific DNA-protein interactions provide a complement to structural studies when trying to understand the molecular basis for sequence specificity. We have used fluorescence spectroscopy to study the chemical equilibrium between the wild-type and a triple mutant glucocorticoid receptor DNA-binding domain (GR DBDwt and GR DBDEGA, respectively) and four related DNA-binding sites (response elements). NMR spectroscopy was used to confirm that the structure of the two proteins is very similar in the uncomplexed state. Binding to DNA oligomers containing single halfsites and palindromic binding sites was studied to obtain separate determinations of association constants and cooperativity parameters involved in the dimeric DNA binding. Equilibrium parameters were determined at 10-35 °C in 85 mM NaCl, 100 mM KC1, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 (20 °C) and at low concentrations of an antioxidant and a nonionic detergent. GR DBDwt binds preferentially to a palindromic consensus glucocorticoid response element (GRE) with an association constant of (7.6 ± 0.9) × 105 M-1 and a cooperativity parameter of 10 ± 1 at 20 °C. GR DBDEGA has the highest affinity for an estrogen response element (ERE) with an association constant of (2.2 ±0.3) × 105 M-1 and a cooperativity parameter of 121 ± 17 at 20 °C. The difference in cooperativity in the two binding processes, which indicates significant differences in binding modes, was confirmed using gel mobility shift assays, van't Hoff analysis shows that DNA binding in all cases is entropy driven within the investigated temperature range. We find that ΔH°obs and Δ5°obs for the formation of a GR DBDwt,-GRE versus GR DBDEGA-ERE complex are significantly different despite very similar ΔG°obs values. A comparison of GR DBDwt, binding to two similar GREs reveals that the discrimination between these two (specific) sites is due to a favorable A(Δ5°obs) which overcompensates an unfavorable Δ(ΔH°obs), i-e., the sequence specificity is in this case entropy driven. Thus, entropie effects are of decisive importance for the affinity as well as the specificity in GR-DNA interactions. The molecular basis for measured equilibrium and thermodynamic parameters is discussed on the basis of published structures of GR DBD-GRE and ER DBD-ERE complexes.

Original languageEnglish (US)
Pages (from-to)5955-5965
Number of pages11
JournalBiochemistry
Volume33
Issue number19
DOIs
StatePublished - May 1 1994

ASJC Scopus subject areas

  • Biochemistry

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