Bradshaw J M, Grucza R A, Ladbury J E, Waksman G
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Biochemistry. 1998 Jun 23;37(25):9083-90. doi: 10.1021/bi973147k.
Src homology 2 (SH2) domains are protein modules that specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two-pronged plug two-holed socket" mechanism whereby binding is hypothesized to resemble a two-pronged plug (the peptide) inserting into a two-holed socket (the SH2 domain). This binding model predicts (1) a hydrophobic basis for high-affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and (2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (Delta G degrees, Delta H degrees, Delta S degrees, Delta Cp degrees) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides that have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism that does not involve substantial conformational changes, the Delta Cp degrees values for all peptides were small and, at least for the high-affinity interactions, similar to the Delta Cp degrees values predicted from surface area calculations. However, unexpectedly, this study reveals that high-affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the Delta Cp degrees values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two-pronged plug two-holed socket" model is an oversimplification of the Src SH2 domain binding mechanism.
Src同源2（SH2）结构域是一种蛋白质模块，可特异性结合信号蛋白上的酪氨酸磷酸化肽段。对Src激酶SH2结构域的X射线晶体学研究探究了其结合机制，从而得出了“双叉插头双孔插座”机制，据此推测结合过程类似于一个双叉插头（肽段）插入一个双孔插座（SH2结构域）。该结合模型预测：（1）高亲和力结合的疏水基础很大程度上由肽段中磷酸酪氨酸C端第三个残基插入SH2结构域主要疏水口袋（+3结合口袋）的程度决定；（2）结合机制涉及SH2结构域无显著构象变化。在本研究中，我们通过等温滴定量热法探究了这些预测，以获取Src SH2结构域与两个系列酪氨酸磷酸肽结合的完整热力学曲线（ΔG°、ΔH°、ΔS°、ΔCp°）。一个系列由经X射线晶体学测定肽段+3位插入+3结合口袋程度不同的肽段组成。另一个系列由在+3位具有逐渐变小的疏水侧链（异亮氨酸、亮氨酸、缬氨酸和丙氨酸）的肽段组成。与不涉及大量构象变化的结合机制一致，所有肽段的ΔCp°值都很小，至少对于高亲和力相互作用而言，类似于根据表面积计算预测的ΔCp°值。然而，出乎意料的是，本研究表明高亲和力结合仅部分由肽段中+3残基与+3结合口袋之间的相互作用决定。此外，所研究的所有肽段的ΔCp°值都相似，这意味着结合时+3结合口袋的去溶剂化程度相似。这些结果表明，“双叉插头双孔插座”模型对Src SH2结构域结合机制的描述过于简单。
