The Vnd/nk-2 Homeodomain Stability And DNA Binding

同源结构域是一类蛋白质的高度保守的DNA结合域，其功能是转录调节，在胚胎细胞对特定发育途径的承诺中指定位置和时间信息。与发育相关的一系列事件的早期步骤是转录调节因子(含有同源结构域的蛋白质)与特定的DNA序列或一组特定的DNA序列的序列特异性结合。在过去三年中研究并于2001年发表在《生物化学》杂志上的同源结构域由？VND？(腹侧神经系统缺陷)/NK-2基因，是由Kim和Nirenberg首先描述的VND/NK-2同源结构域的亲代成员。[关于NKX-2.5同源结构域蛋白的研究已经启动，但结果是初步的，这里没有报道。NKX-2.5同源结构域蛋白是人类心脏发育早期必不可少的转录调节因子。]用差示扫描量热法(DSC)和222 nm处的椭圆度变化研究了VND/NK-2同源结构域[HD(Wt)；包含60个残基的1-80个残基]和DNA识别位点螺旋III[HD(H52R)和HD(H52R/T56W)]的构象稳定性。在50-500 mM的氯化钠存在下，p=0.52+/-0.04kcal/KmoL，pH为7.4的热去折叠反应是可逆的和可重复的。50 mM磷酸盐或100-500 mM NaC l加入50 mM HEPES pH 7.4缓冲液(从35.5℃转变到43-51℃，范特霍夫热变47+/-5千卡/摩尔)可使HD(Wt)稳定。稳定性顺序为HD(H52R/T56W)&GT；HD(H52R)&GT；HD(Wt)，与存在的阴离子无关。用两态去折叠模型很好地拟合了222 nm处椭圆度随温度升高的变化曲线，突变的同源结构域的二级结构变化的协同性大于HD(Wt)，并且在Hepes缓冲液中加入100 mM的NaC l也增加了二级结构变化的协同性。磷酸盐结合对HD(Wt)固有色氨酸残基荧光的33%猝灭(估计磷酸盐的解离常数约为2.6 mm)被DNA结合逆转了60%。用等温滴定量热法(10-30℃)测定了VND/NK-2同源域蛋白与序列特异性18个碱基DNA结合的热力学参数。HD(Wt)、HD(H52R)和HD(H52R/T56W)结合双链DNA的热容变化值分别为+0.25、-0.17和-0.10+/-0.04千卡/千克摩尔。同源结构域与DNA的相互作用在298K、pH 7.4时热变为-6.6+/-0.5、-10.8+/-0.1和-9.0+/-0.6kcal/mol，吉布斯自由能变化为-11.0+/-0.1、-11.0+/-0.1和-11.3+/-0.3kcal/mol，结合化学计量比为1.0+/-0.1。DNA结合的热力学参数不能从与DNA络合时发生的同源结构域结构变化中预测出来，因此也必须反映溶剂和可能的DNA重排。

The homeodomain is the highly conserved DNA-binding domain of a class of proteins that function as transcriptional regulators, specifying positional and temporal information in the commitment of embryonic cells to specific developmental pathways. An early step in the cascade of events associated with development is the sequence-specific binding of the transcriptional regulator (the homeodomain-containing protein) to a specific sequence or a specific set of sequences of DNA. The homeodomain studied in the last three years and published in Biochemistry in 2001 is encoded by the ?vnd? (ventral nervous system defective)/NK-2 gene of Drosophila melanogaster and is the parent member of the vnd/NK-2 class of homeodomains first described by Kim and Nirenberg. [Studies on the NKX-2.5 homeodomain protein, an essential transcriptional regulator in early human heart development, have been initiated but results are preliminary and are not reported here.] The conformational stabilities of the vnd/NK-2 homeodomain [HD(wt); residues 1-80 that encompasses the 60-residue homeodomain] and those harboring mutations in helix III of the DNA recognition site [HD(H52R) and HD(H52R/T56W)] have been investigated by differential scanning calorimetry (DSC) and ellipticity changes at 222 nm. Thermal unfolding reactions at pH 7.4 are reversible and repeatable in the presence of 50-500 mM NaCl with delta Cp = 0.52 +/- 0.04 kcal/K mol. A substantial stabilization of HD(wt) is produced by 50 mM phosphate or by the addition of 100-500 mM NaCl to 50 mM Hepes pH 7.4 buffer (from a transition temperature of 35.5 to 43 - 51 C with van?t Hoff enthalpy changes of 47 +/- 5 kcal/mol). The order of stability is HD(H52R/T56W) > HD(H52R) > HD(wt), irrespective of the anions present. Progress curves for ellipticity changes at 222 nm as a function of increasing temperature are fitted well by a two-state unfolding model, and the cooperativity of secondary structure changes is greater for mutant homeodomains than for HD(wt) and also is increased by adding 100 mM NaCl to Hepes buffer. A 33% quench of the intrinsic tryptophanyl residue fluorescence of HD(wt) by phosphate binding (with a dissociation constant of ca. 2.6 mM estimated for phosphate) is reversed 60% by DNA binding. Thermodynamic parameters for vnd/NK-2 homeodomain proteins binding sequence-specific 18 bp DNA have been determined by isothermal titration calorimetry (10 - 30 C). Values of heat capacity changes are +0.25, -0.17, and -0.10 +/- 0.04 kcal/K mol for HD(wt), HD(H52R), and HD(H52R/T56W) binding duplex DNA, respectively. Interactions of homeodomains with DNA are enthalpically controlled at 298 K and pH 7.4 with corresponding enthalpy changes of -6.6 +/- 0.5, -10.8 +/- 0.1, and -9.0 +/-0.6 kcal/mol and Gibbs free energy changes of -11.0 +/- 0.1, -11.0 +/- 0.1, and -11.3 +/- 0.3 kcal/mol with a binding stoichiometry of 1.0 +/- 0.1. Thermodynamic parameters for DNA binding are not predicted from homeodomain structural changes that occur upon complexing to DNA and therefore must reflect also solvent and possibly DNA rearrangements.