THERMODYNAMICS OF SEQUENCE-SPECIFIC GLUCOCORTICOID RECEPTOR DNA INTERACTIONS

The thermodynamics of sequence-specific DNA-protein interactions provi de a complement to structural studies when trying to understand the mo lecular basis for sequence specificity. We have used fluorescence spec troscopy to study the chemical equilibrium between the wild-type and a triple mutant glucocorticoid receptor DNA-binding domain (GR DBDwt an d GR DBDEGA, respectively) and four related DNA-binding sites (respons e elements). NMR spectroscopy was used to confirm that the structure o f the two proteins is very similar in the uncomplexed state. Binding t o DNA oligomers containing single half-sites and palindromic binding s ites was studied to obtain separate determinations of association cons tants and cooperativity parameters involved in the dimeric DNA binding . Equilibrium parameters were determined at 10-35 degrees C in 85 mM N aCl, 100 mM KCl, 2 mM MgCl2, and 20 mM Tris-HCl at pH 7.4 (20 degrees C) and at low concentrations of an antioxidant and a nonionic detergen t. GR DBDwt binds preferentially to a palindromic consensus glucocorti coid response element (GRE) with an association constant of (7.6 +/- 0 .9) x 10(5) M(-1) and a cooperativity parameter of 10 +/- 1 at 20 degr ees C. GR DBDEGA has the highest affinity for an estrogen response ele ment (ERE) with an association constant of(2.2 +/- 0.3) x 10(5) M(-1) and a cooperativity parameter of 121 +/- 17 at 20 degrees C. The diffe rence in cooperativity in the two binding processes, which indicates s ignificant differences in binding modes, was confirmed using gel mobil ity shift assays. van't Hoff analysis shows that DNA binding in all ca ses is entropy driven within the investigated temperature range. We fi nd that Delta H degrees(obs) and Delta S degrees(obs) for the formatio n of a GR DBDwt-GRE versus GR DBDEGA-ERE complex are significantly dif ferent despite very similar Delta G degrees(obs) values. A comparison of GR DBDwt binding to two similar GREs reveals that the discriminatio n between these two (specific) sites is due to a favorable Delta(Delta S degrees(obs)) which overcompensates an unfavorable Delta(Delta H de grees(obs)), i.e., the sequence specificity is in this case entropy dr iven. Thus, entropic effects are of decisive importance for the affini ty as well as the specificity in GR-DNA interactions. The molecular ba sis for measured equilibrium and thermodynamic parameters is discussed on the basis of published structures of GR DBD-GRE and ER DBD-ERE com plexes.