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结合结构域，其作为转录调节因子起作用，在胚胎细胞向特定发育途径的定向中指定位置和时间信息。与发育相关的级联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序列组的序列特异性结合。同源结构域的研究在过去的三年中，并发表在生物化学在2001年编码的？vnd？（腹神经系统缺陷）/NK-2基因，并且是由Kim和Nirenberg首先描述的vnd/NK-2类同源结构域的亲本成员。[对NKX-2.5同源结构域蛋白（人类心脏发育早期的重要转录调节因子）的研究已经开始，但结果是初步的，这里没有报道。 vnd/NK-2同源结构域[HD（wt）;残基1-80，包括60个残基的同源结构域]和那些在DNA识别位点的螺旋III中具有突变的构象稳定性[HD（H52 R）和HD（H52 R/T56 W）]已经通过差示扫描量热法（DSC）和在222 nm处的椭圆度变化进行了研究。在50-500 mM NaCl存在下，pH 7.4下的热去折叠反应是可逆和可重复的，Δ Cp = 0.52 +/- 0.04 kcal/K mol。通过50 mM磷酸盐或通过向50 mM Hepes pH 7.4缓冲液中加入100-500 mM NaCl（用货车？t霍夫焓变为47 +/-5千卡/摩尔）。其稳定性顺序为HD（H_（52）R/T_（56）W）> HD（H_（52）R）> HD（wt），与存在的阴离子无关。在222 nm处的椭圆度变化作为温度升高的函数的进展曲线拟合良好的两态展开模型，和二级结构变化的协同性是更大的突变体同源结构域比HD（重量），也增加了通过添加100 mM NaCl Hepes缓冲液。HD（wt）的内源性β-羟基残基荧光通过磷酸盐结合（解离常数约为100%）猝灭33%。2.6磷酸盐估计的mM）被DNA结合逆转60%。已通过等温滴定量热法（10 - 30 ℃）测定了vnd/NK-2同源域蛋白结合序列特异性18 bp DNA的热力学参数。对于HD（wt）、HD（H52 R）和HD（H52 R/T56 W）结合双链体DNA，热容变化值分别为+0.25、-0.17和-0.10 +/-0.04 kcal/K mol。同源结构域与DNA的相互作用在298 K和pH 7.4下受热力学控制，相应的焓变为-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、-1.0 +/-0.1和-11.3 +/-0.3千卡/摩尔，结合化学计量为1.0 +/-0.1。DNA结合的热力学参数不能从与DNA络合后发生的同源结构域结构变化中预测，因此必须也反映溶剂和可能的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.