Department of Medicinal Chemistry, Markey Center for Structural Biology, and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.
J Am Chem Soc. 2010 Aug 18;132(32):11058-70. doi: 10.1021/ja910535j.
NMR spectroscopy and molecular dynamics (MD) simulations were used to probe the structure and dynamics of complexes of three phosphotyrosine-derived peptides with the Src SH2 domain in an effort to uncover a structural explanation for enthalpy-entropy compensation observed in the binding thermodynamics. The series of phosphotyrosine peptide derivatives comprises the natural pYEEI Src SH2 ligand, a constrained mimic, in which the phosphotyrosine (pY) residue is preorganized in the bound conformation for the purpose of gaining an entropic advantage to binding, and a flexible analogue of the constrained mimic. The expected gain in binding entropy of the constrained mimic was realized; however, a balancing loss in binding enthalpy was also observed that could not be rationalized from the crystallographic structures. We examined protein dynamics to evaluate whether the observed enthalpic penalty might be the result of effects arising from altered motions in the complex. (15)N-relaxation studies and positional fluctuations from molecular dynamics indicate that the main-chain dynamics of the protein show little variation among the three complexes. Root mean squared (rms) coordinate deviations vary by less than 1.5 A for all non-hydrogen atoms for the crystal structures and in the ensemble average structures calculated from the simulations. In contrast to this striking similarity in the structures and dynamics, there are a number of large chemical shift differences from residues across the binding interface, but particularly from key Src SH2 residues that interact with pY, the "hot spot" residue, which contributes about one-half of the binding free energy. Rank-order correlations between chemical shifts and ligand binding enthalpy for several pY-binding residues, coupled with available mutagenesis and calorimetric data, suggest that subtle structural perturbations (<1 A) from the conformational constraint of the pY residue sufficiently alter the geometry of enthalpically critical interactions in the binding pocket to cause the loss of binding enthalpy, leading to the observed enthalpy-entropy compensation. We find no evidence to support the premise that enthalpy-entropy compensation is an inherent property and conclude that preorganization of Src SH2 ligand residues involved in binding hot spots may eventuate in suboptimal interactions with the domain. We propose that introducing constraints elsewhere in the ligand could minimize enthalpy-entropy compensation effects. The results illustrate the utility of the NMR chemical shift to highlight small, but energetically significant, perturbations in structure that might otherwise go unnoticed in an apparently rigid protein.
NMR 光谱和分子动力学（MD）模拟被用于探测三个磷酸酪氨酸衍生肽与Src SH2 结构域的复合物的结构和动力学，以揭示在结合热力学中观察到的焓熵补偿的结构解释。该系列磷酸酪氨酸肽衍生物包括天然 pYEEI Src SH2 配体、一种约束模拟物，其中磷酸酪氨酸（pY）残基在结合构象中预先组织，目的是获得结合的熵优势，以及约束模拟物的柔性类似物。约束模拟物的结合熵预期增加得到了实现；然而，也观察到结合焓的平衡损失，这无法从晶体结构中得到合理化。我们检查了蛋白质动力学，以评估观察到的焓罚是否可能是由于复合物中运动改变而产生的影响。（15）N 弛豫研究和分子动力学的位置波动表明，三种复合物之间蛋白质的主链动力学变化很小。所有非氢原子的均方根（rms）坐标偏差在晶体结构和模拟计算的平均结构中变化小于 1.5 A。与结构和动力学的这种惊人相似性形成对比的是，结合界面上的许多残基存在较大的化学位移差异，但特别是与与 pY 相互作用的关键 Src SH2 残基的化学位移差异较大，pY 是“热点”残基，贡献了约一半的结合自由能。几个 pY 结合残基的化学位移与配体结合焓之间的秩相关，加上可用的诱变和量热数据，表明 pY 残基的构象约束所引起的微小结构扰动（<1 A）足以改变结合口袋中焓关键相互作用的几何形状，导致结合焓的损失，导致观察到的焓熵补偿。我们没有发现证据支持焓熵补偿是固有特性的前提，并得出结论，参与结合热点的 Src SH2 配体残基的预组织可能导致与该结构域的次优相互作用。我们提出，在配体的其他地方引入约束可以最小化焓熵补偿效应。结果表明，NMR 化学位移可用于突出结构中的小但能量重要的扰动，否则在明显刚性的蛋白质中可能会被忽略。
