Thermodynamics of sequence-specific glucocorticoid receptor-DNA interactions

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 DBD wt 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 half-sites 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 degrees C in 85 mM NaCl, 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 detergent. GR DBDwt binds preferentially to a palindromic consensus glucocorticoid 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 degrees C. GR DBDEGA has the highest affinity for an estrogen response element (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 difference in cooperativity in the two binding processes, which indicates significant differences in binding modes, was confirmed using gel mobility assays. van't Hoff analysis shows that DNA binding in all cases in entropy driven within the investigated temperature range. We find that delta H0obs and delta S0obs for the formation of a GR DBDwt-GRE versus GR DBDEGA-ERE complex are significantly different despite very similar delta G0obs 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 delta(delta S0obs) which overcompensates an unfavorable delta(delta H0obs), i.e., the sequence specificity is in this case entropy driven. Thus, entropic 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.

Sequence-specific DNA-binding dominated by dehydration.

“…The wild-type GR DBD (equal to the fragment K438-Q520 of the rat GR and the fragment K419-Q501 of the human GR) was overexpressed in Escherichia coli and purified as described (13 tometrically using the extinction coefficient 6280 nm = 4200 M-1*cm-1 calculated for tyrosine absorption (22). DNA Preparation.…”

“…Fluorescence quenching experiments were performed as reverse titrations, in which different amounts of DNA were added at a constant protein concentration. The titration procedure including experimental set-up, fluorescence excitation and emission wavelengths, corrections for background fluorescence, light scattering and optical filtering effects, and precautions to photochemical degradation has been described previously (13). Buffer A (without DTT) was carefully degassed by stirring the buffer while applying vacuum before DTT (1 mM) and GR DBD (2 ,tM) were added.…”

“…The DNA was then added in 20 subsequent 1-,ul aliquots. Concentrations of free and bound protein were calculated from the fractional fluorescence quenching and equilibrium parameters were determined by nonlinear least square fits of theoretical binding isotherms to data representing the mean of 2-4 independent titrations (13).…”

“…The DBDs bind as homodimers to the response elements, which are partially palindromic DNA sequences, with the recognition helix of each protein monomer -placed in adjacent major grooves on DNA. We previously studied the binding of native and mutant GR DBDs to DNA using equilibrium titrations based on fluorescence spectroscopy (13,19,20). The thermodynamics of complex formation investigated by van't Hoff analyses revealed unfavorable binding enthalpies at low temperatures (i.e., entropy-driven reactions) and negative changes in heat capacity, suggesting contributions from dehydration of interacting surfaces.…”

“…A consequence of this is that the binding is entropy driven at low temperatures. Moreover, the discrimination between different specific binding sites (13) as well as between specific and nonspecific binding sites (14) can also be entropy driven. Therefore, it is necessary to combine structural studies with detailed thermodynamic analyses to understand the physical basis for sequence specificity (15).…”