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

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• 作者：Richard A. Grucza ; J. Michael Bradshaw ; Vesselin Mitaxov ; and Gabriel Waksman
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：August 22, 2000
• 年：2000
• 卷：39
• 期：33
• 页码：10072 - 10081
• 全文大小：238K
• 年卷期：v.39,no.33(August 22, 2000)
• ISSN：1520-4995

文摘

SH2 domains are small protein domains that bind specifically to tyrosyl-phosphorylatedsequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulatedthat 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 andSH2 domains. The dependence of the binding constant, K_obs, on [NaCl] was shown to be strong for bindingof the tandem SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptides derivedfrom 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 ofthe Src kinase to its consensus singly phosphorylated peptide (sequence pYEEI where pY indicates aphosphotyrosine) was also highly dependent on [NaCl] with a SK_obs value of -2.4 ± 0.1. The ability ofsalt to disrupt the interactions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependentwith the inhibitory effect following the order: phosphate > Cl^- > F^-. For the Syk-tSH2 system, interactionsin 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% andweakened binding by 2-3 orders of magnitude. Consistent with this finding, binding of the single aminoacid Ac-pY-NH₂ was characterized by a large salt dependence of binding and was also dependent on theidentity of the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicatedin determining the specificity of the phosphopeptide-SH2 domain interaction, was next probed bycomparing the binding of the Src SH2 domain to a peptide containing the pYEEI sequence with that ofa 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, residuesconferring 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 recognitionand binding but also in contributing to the specificity of the interactions between tyrosyl phosphopeptidesand SH2 domains.