The Vnd/nk-2 Homeodomain Stability And DNA Binding

同源结构域是一类高度保守的dna结合结构域，其功能是转录调控，在胚胎细胞向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（含同源结构域的蛋白质）与特定序列或特定DNA序列集的序列特异性结合。在过去三年研究并于2001年发表在《生物化学》上的同位域是由？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类同源结构域的亲本成员。[NKX-2.5同源结构域蛋白是早期人类心脏发育的重要转录调节因子，研究已经开始，但结果是初步的，这里没有报道。]vnd/NK-2同域的构象稳定性[HD(wt)]；利用差示扫描量热法（DSC）和222 nm的椭圆度变化研究了包含60个同位结构域的残基1-80和DNA识别位点螺旋III突变的残基[HD（H52R）和HD(H52R/T56W)]。pH 7.4下的热展开反应在50-500 mM NaCl （δ Cp = 0.52 +/- 0.04 kcal/K mol）存在下是可逆且可重复的。通过50 mM磷酸盐或在50 mM Hepes pH 7.4缓冲液中加入100-500 mM NaCl（从35.5℃过渡到43 - 51℃），可以产生大量的HD（wt）稳定化。霍夫焓变为47±5千卡/摩尔)。稳定性的顺序为HD(H52R/T56W) > HD(H52R) > HD(wt)，而不考虑阴离子的存在。两态展开模型拟合了222 nm下椭圆度随温度变化的进展曲线，突变体同源结构域的二级结构变化协同性高于HD(wt)，并且在Hepes缓冲液中加入100 mM NaCl后二级结构变化协同性也有所提高。磷酸盐结合（估计磷酸盐的解离常数约为2.6 mM）使HD（wt）固有色氨酸残基荧光猝灭33%，而DNA结合则使其猝灭60%。用等温滴定量热法（10 - 30℃）测定了vnd/NK-2同源结构域蛋白结合序列特异性18bp DNA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 kcal/K mol。同位结构域与DNA的相互作用焓控制在298 K和pH 7.4，对应的焓变为-6.6 +/- 0.5、-10.8 +/- 0.1和-9.0 +/-0.6 kcal/mol，吉布斯自由能变化为-11.0 +/- 0.1、-11.0 +/- 0.1和-11.3 +/- 0.3 kcal/mol，结合化学计量量为1.0 +/- 0.1。DNA结合的热力学参数不能通过与DNA络合时发生的同域结构变化来预测，因此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.