Role of electrostatic interactions in SH2 domain recognition: Salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase
Role of electrostatic interactions in SH2 domain recognition: Salt-dependence of tyrosyl-phosphorylated peptide binding to the tandem SH2 domain of the Syk kinase and the single SH2 domain of the Src kinase
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DOI：
10.1021/bi000891n
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发表时间：
2000-08-22
期刊：
影响因子：
2.9
通讯作者：
Waksman, G
中科院分区：
文献类型：
--
作者：
Grucza, RA;Bradshaw, JM;Waksman, G
Sh2结构域是一种小的蛋白质结构域，能与酪氨酸磷酸化的序列特异性结合。由于磷酸化贡献了结合自由能的很大一部分，因此推测静电相互作用可能在SH2结构域的识别中发挥重要作用。为了验证这一假设，我们研究了酪氨酸磷酸化肽和SH2结构域之间相互作用的盐依赖性。对于Syk-tSH2结构域(Syk-tSH2)与来自免疫受体酪氨酸激活基序(DpITAM)的双磷酸化多肽(DpITAM)的结合，结合常数K-OBS对[氯化钠]的依赖性很强：LOG(K-OBS)与LOG[氯化钠]的曲线图斜率在-2.6+/-0.1到-3.1+/-0.2之间。Src激酶的单个SH2结构域与其共同的单磷酸化肽(序列pYEEI，其中Py表示磷酸化酪氨酸)的结合也高度依赖于Sko(Bs)值为-2.4+/-0.1的[氯化钠]。盐对Syk-tSH2和dpITAM多肽相互作用的破坏作用是阴离子依赖性的，其抑制作用顺序为：磷酸>；Cl->；F-。对于Syk-tSEI2系统，Py结合口袋中的相互作用被证明是导致总盐依赖性的很大一部分原因：从dpITAM多肽中去除任何一种磷酸盐都会使SKOB的数量减少40-60%，结合能力减弱2-3个数量级。与这一发现一致的是，单一氨基酸Ac-Py-NH2的结合具有很大的结合盐依赖性，并且还依赖于扰动阴离子的同一性。接下来，通过比较Src SH2结构域与包含pYEEI序列的肽与低亲和力变异体pYAAI肽的结合：与pYEEI肽的-2.4+/-0.1相比，变异肽的SKOB的大小降低到-1.3+/-0.1，从而探索了肽残基对Py的作用，这在确定磷酸肽-SH2结构域相互作用的特异性中是可能的，这表明除了Py，研究表明，静电相互作用不仅在介导Py的识别和结合中起重要作用，而且在酪氨酸磷酸肽与SH2结构域相互作用的专一性中也起重要作用。
SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interactions may play an important role in SH2 domain recognition. To test this hypothesis, we have examined the salt dependence of the interaction between tyrosyl-phosphorylated peptides and SH2 domains. The dependence of the binding constant, K-obs, On [NaCl] was shown to be strong for binding of the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derived from immune-receptor tyrosine activation motifs (dpITAMs): the slopes of plots of log(K-obs) versus log [NaCl], designated SKobs, ranged from -2.6 +/- 0.1 to -3.1 +/- 0.2. Binding of the single SH2 domain of the Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates a phosphotyrosine) was also highly dependent on [NaCl] with a SKo(bs) value of -2.4 +/- 0.1. The ability of salt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl- > F-. For the Syk-tSEI2 system, interactions in the pY-binding pockets were shown to be responsible for a large portion of the total salt dependence: removal of either phosphate from the dpITAM peptide reduced the magnitude of SKobs by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH2 was characterized by a large salt dependence of binding and was also dependent on the identity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed by comparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that of a lower affinity variant pYAAI peptide: the magnitude of SKobs for the variant peptide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI peptide, indicating that in addition to pY, residues conferring peptide binding specificity contribute significantly to the salt dependence of SH2 domain binding This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.