Lundbäck T, Zilliacus J, Gustafsson J A, Carlstedt-Duke J, Härd T
Center for Structural Biochemistry, Karolinska Institutet, Huddinge, Sweden.
Biochemistry. 1994 May 17;33(19):5955-65. doi: 10.1021/bi00185a037.
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.
在试图理解序列特异性的分子基础时，序列特异性DNA - 蛋白质相互作用的热力学为结构研究提供了补充。我们利用荧光光谱研究了野生型和三重突变糖皮质激素受体DNA结合结构域（分别为GR DBD wt和GR DBDEGA）与四个相关DNA结合位点（反应元件）之间的化学平衡。核磁共振光谱用于确认两种蛋白质在未结合状态下的结构非常相似。研究了与含有单个半位点和回文结合位点的DNA寡聚物的结合，以分别测定二聚体DNA结合中涉及的缔合常数和协同参数。在85 mM NaCl、100 mM KCl、2 mM MgCl2和20 mM Tris - HCl（pH 7.4，20℃）以及低浓度抗氧化剂和非离子去污剂存在的条件下，于10 - 35℃测定平衡参数。GR DBDwt优先结合回文共有糖皮质激素反应元件（GRE），在20℃时缔合常数为(7.6±0.9)×10⁵ M⁻¹，协同参数为10±1。GR DBDEGA对雌激素反应元件（ERE）具有最高亲和力，在20℃时缔合常数为(2.2±0.3)×10⁵ M⁻¹，协同参数为121±17。两种结合过程中协同性的差异表明结合模式存在显著差异，这通过凝胶迁移试验得到了证实。范特霍夫分析表明，在所研究的温度范围内，所有情况下的DNA结合都是由熵驱动的。我们发现，尽管GR DBDwt - GRE与GR DBDEGA - ERE复合物形成的ΔG0obs值非常相似，但ΔH0obs和ΔS0obs却有显著差异。GR DBDwt与两个相似GRE结合的比较表明，这两个（特异性）位点之间的区分是由于有利的Δ(ΔS0obs)，它补偿了不利的Δ(ΔH0obs)，即这种情况下序列特异性是由熵驱动的。因此，熵效应对于GR - DNA相互作用中的亲和力和特异性起着决定性作用。基于已发表的GR DBD - GRE和ER DBD - ERE复合物结构，讨论了所测平衡和热力学参数的分子基础。
