Berger C, Jelesarov I, Bosshard H R
Biochemisches Institut, Universität Zürich, Switzerland.
Biochemistry. 1996 Nov 26;35(47):14984-91. doi: 10.1021/bi961312a.
The site-specific interaction of the basic leucine zipper protein C62GCN4, which corresponds to the C-terminal sequence 220-281 of the yeast transcription factor GCN4, with the AP-1 and ATF/ CREB DNA recognition sites was analyzed by isothermal titration microcalorimetry. Free C62GCN4 is a dimer composed of a C-terminal leucine zipper and a basic, mainly unstructured DNA binding domain. Upon association with the target DNA, C62GCN4 folds to a fully alpha-helical dimer [Ellenberger et al. (1992) Cell 71, 1223-1237; König and Richmond (1993) J. Mol. Biol. 233, 139-154]. The protein-bound AP-1 site is straight, and the protein-bound ATF/CREB site is bent by 20 degrees toward the leucine zipper domain. The coupling between protein folding and DNA association resulting in two conformationally different complexes with C62GCN4 poses interesting thermodynamic problems. The association was strongly exothermic for both DNA target sites. The free energies of binding were indistinguishable in buffers of low salt concentration, and no change of the protonation state of C62GCN4 and/or the DNA target site occurred on formation of the complexes. Both complexes exhibited large and negative heat capacity changes. The empirical correlation between buried nonpolar and polar surfaces and the reduction in heat capacity concomitant to complexation did hold for the reaction with the AP-1 site at low salt concentration. However, in the case of the ATF/CREB site, the change in heat capacity was larger than could be accounted for by the burial of solvent-accessible surface. Potential sources of the extra decrement in the heat capacity could be restrictions in the vibrational modes of polar groups and of bound water molecules at the protein-DNA interface, thought to result from the bending of the ATF/CREB site. In the presence of high concentrations of glutamate and NaCl, the complex with the ATF/CREB site was significantly weaker than the complex with the AP-1 site.
通过等温滴定量热法分析了碱性亮氨酸拉链蛋白C62GCN4（对应于酵母转录因子GCN4的C末端序列220 - 281）与AP - 1和ATF/CREB DNA识别位点的位点特异性相互作用。游离的C62GCN4是由一个C末端亮氨酸拉链和一个碱性的、主要为无结构的DNA结合结构域组成的二聚体。与靶DNA结合后，C62GCN4折叠成一个完全α螺旋的二聚体[埃伦伯格等人（1992年）《细胞》71卷，1223 - 1237页；柯尼希和里士满（1993年）《分子生物学杂志》233卷，139 - 154页]。与蛋白质结合的AP - 1位点是直的，而与蛋白质结合的ATF/CREB位点朝着亮氨酸拉链结构域弯曲了20度。蛋白质折叠与DNA结合之间的耦合导致了与C62GCN4形成两种构象不同的复合物，这带来了有趣的热力学问题。对于两个DNA靶位点，结合都是强烈放热的。在低盐浓度缓冲液中，结合自由能没有区别，并且在复合物形成过程中C62GCN4和/或DNA靶位点的质子化状态没有变化。两种复合物都表现出大的负热容变化。在低盐浓度下，对于与AP - 1位点的反应，埋藏的非极性和极性表面与复合物形成时伴随的热容降低之间的经验相关性确实成立。然而，在ATF/CREB位点的情况下，热容变化比可由溶剂可及表面的埋藏所解释的要大。热容额外降低的潜在来源可能是蛋白质 - DNA界面处极性基团和结合水分子振动模式的限制，据认为这是由ATF/CREB位点的弯曲导致的。在高浓度谷氨酸和氯化钠存在的情况下，与ATF/CREB位点形成的复合物比与AP - 1位点形成的复合物明显弱。
