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首页> 外文期刊>Biochemistry >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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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.

机译：静电相互作用在SH2结构域识别中的作用：酪氨酸磷酸化肽与Syk激酶的串联SH2结构域和Src激酶的单个SH2结构域结合的盐依赖性。

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摘要

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 SK(obs), 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 SK(obs) 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-tSH2 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 SK(obs) by 40-60% and weakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single amino acid Ac-pY-NH(2) 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 SK(obs) 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.
机译：SH2结构域是特异性结合酪氨酰磷酸化序列的小蛋白结构域。由于磷酸化贡献了大部分的结合自由能，因此推测静电相互作用可能在SH2域识别中起重要作用。为了检验该假设，我们检查了酪氨酰磷酸化肽和SH2结构域之间相互作用的盐依赖性。结合常数K（obs）对[NaCl]的依赖性显示出对于Syk激酶的串联SH2域（Syk-tSH2）与源自免疫受体酪氨酸激活基序的双磷酸化肽结合的强烈依赖性（ dpITAMs）：log（K（obs））与log [NaCl]的曲线斜率，称为SK（obs），范围为-2.6 +/- 0.1至-3.1 +/- 0.2。 Src激酶的单个SH2结构域与其共有的单磷酸化肽段（序列pYEEI，其中pY表示磷酸酪氨酸）的结合也高度依赖于[NaCl]，SK（obs）值为-2.4 +/- 0.1。盐破坏Syk-tSH2与dpITAM肽之间相互作用的能力显示为阴离子依赖性，其抑制作用的顺序为：磷酸盐> Cl（-）> F（-）。对于Syk-tSH2系统，显示pY结合口袋中的相互作用是总盐依赖性的很大部分：从dpITAM肽中除去任何一种磷酸盐均可将SK（obs）的幅度降低40-60％并减弱了2-3个数量级的结合力。与该发现一致，单个氨基酸Ac-pY-NH（2）的结合特征是结合的盐依赖性大，并且还取决于干扰阴离子的身份。接下来，通过比较Src SH2结构域与含有pYEEI序列的肽与pYEEI序列的结合，来探究pY的C末端肽残基的作用，这与确定磷酸肽-SH2结构域相互作用的特异性有关。亲和力较低的变体pYAAI肽：相对于pYEEI肽的-2.4 +/- 0.1，变体肽的SK（obs）值降低至-1.3 +/- 0.1，这表明除pY外，残基还具有肽结合特异性显着促进了SH2域结合的盐依赖性。这项研究表明，静电相互作用不仅在介导pY识别和结合中起重要作用，而且在酪氨酸磷酸肽和SH2结构域之间相互作用的特异性贡献中也起着重要作用。

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