Casares Salvador, Ab Eiso, Eshuis Henk, Lopez-Mayorga Obdulio, van Nuland Nico A J, Conejero-Lara Francisco
Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, Granada, Spain.
BMC Struct Biol. 2007 Apr 2;7:22. doi: 10.1186/1472-6807-7-22.
SH3 domains are small protein modules of 60-85 amino acids that bind to short proline-rich sequences with moderate-to-low affinity and specificity. Interactions with SH3 domains play a crucial role in regulation of many cellular processes (some are related to cancer and AIDS) and have thus been interesting targets in drug design. The decapeptide APSYSPPPPP (p41) binds with relatively high affinity to the SH3 domain of the Abl tyrosine kinase (Abl-SH3), while it has a 100 times lower affinity for the alpha-spectrin SH3 domain (Spc-SH3).
Here we present the high-resolution structure of the complex between the R21A mutant of Spc-SH3 and p41 derived from NMR data. Thermodynamic parameters of binding of p41 to both WT and R21A Spc-SH3 were measured by a combination of isothermal titration and differential scanning calorimetry. Mutation of arginine 21 to alanine in Spc-SH3 increases 3- to 4-fold the binding affinity for p41 due to elimination at the binding-site interface of the steric clash produced by the longer arginine side chain. Amide hydrogen-deuterium experiments on the free and p41-bound R21A Spc-SH3 domain indicate that binding elicits a strong reduction in the conformational flexibility of the domain. Despite the great differences in the thermodynamic magnitudes of binding, the structure of the R21A Spc-SH3:P41 complex is remarkably similar to that of the Abl-SH3:P41 complex, with only few differences in protein-ligand contacts at the specificity pocket. Using empirical methods for the prediction of binding energetics based on solvent-accessible surface area calculations, the differences in experimental energetics of binding between the two complexes could not be properly explained only on the basis of the structural differences observed between the complexes. We suggest that the experimental differences in binding energetics can be at least partially ascribed to the absence in the R21A Spc-SH3:P41 complex of several buried water molecules, which have been proposed previously to contribute largely to the highly negative enthalpy and entropy of binding in the Abl-SH3:P41 complex.
Based on a deep structural and thermodynamic analysis of a low and high affinity complex of two different SH3 domains with the same ligand p41, we underline the importance of taking into account in any effective strategy of rational design of ligands, factors different from the direct protein-ligand interactions, such as the mediation of interactions by water molecules or the existence of cooperative conformational effects induced by binding.
SH3结构域是由60 - 85个氨基酸组成的小蛋白质模块，能以中低亲和力和特异性结合富含脯氨酸的短序列。与SH3结构域的相互作用在许多细胞过程（有些与癌症和艾滋病相关）的调控中起关键作用，因此一直是药物设计中令人感兴趣的靶点。十肽APSYSPPPPP（p41）与Abl酪氨酸激酶的SH3结构域（Abl - SH3）具有相对较高的亲和力，而对α - 血影蛋白SH3结构域（Spc - SH3）的亲和力则低100倍。
在此，我们根据核磁共振数据展示了Spc - SH3的R21A突变体与p41复合物的高分辨率结构。通过等温滴定量热法和差示扫描量热法相结合，测量了p41与野生型和R21A Spc - SH3的结合热力学参数。Spc - SH3中精氨酸21突变为丙氨酸后，由于较长精氨酸侧链在结合位点界面产生的空间冲突被消除，对p41的结合亲和力提高了3至4倍。对游离和与p41结合的R21A Spc - SH3结构域进行的酰胺氢 - 氘交换实验表明，结合导致该结构域的构象灵活性大幅降低。尽管结合的热力学量存在很大差异，但R21A Spc - SH3:P41复合物的结构与Abl - SH3:P41复合物的结构非常相似，仅在特异性口袋处的蛋白质 - 配体接触上有少数差异。使用基于溶剂可及表面积计算预测结合能的经验方法，仅根据复合物之间观察到的结构差异，无法恰当解释两种复合物结合实验能学上的差异。我们认为，结合能学上的实验差异至少部分可归因于R21A Spc - SH3:P41复合物中缺少几个埋藏的水分子，此前有人提出这些水分子在很大程度上对Abl - SH3:P41复合物结合时高度负的焓和熵有贡献。
基于对两种不同SH3结构域与同一配体p41的低亲和力和高亲和力复合物进行的深入结构和热力学分析，我们强调在任何有效的配体合理设计策略中，都要考虑到不同于直接蛋白质 - 配体相互作用的因素，如水分子介导的相互作用或结合诱导的协同构象效应的存在。
