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

SH2 domains are small protein domains that bind specifically to tyrosyl-pho sphorylated sequences. Because phosphorylation contributes a large part of the binding free energy, it has been postulated that electrostatic interact ions may play an important role in SH2 domain recognition. To test this hyp othesis, we have examined the salt dependence of the interaction between ty rosyl-phosphorylated peptides and SH2 domains. The dependence of the bindin g constant, K-obs, On [NaCl] was shown to be strong for binding of the tand em SH2 domain of the Syk kinase (Syk-tSH2) to doubly phosphorylated peptide s derived from immune-receptor tyrosine activation motifs (dpITAMs): the sl opes of plots of log(K-obs) versus log [NaCl], designated SKobs, ranged fro m -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 wher e 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 interac tions between Syk-tSH2 and dpITAM peptides was shown to be anion-dependent with the inhibitory effect following the order: phosphate > Cl- > F-. For t he Syk-tSEI2 system, interactions in the pY-binding pockets were shown to b e 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 thi s 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 o f the perturbing anion. The role of peptide residues C-terminal to the pY, which are implicated in determining the specificity of the phosphopeptide-S H2 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 pept ide was reduced to -1.3 +/- 0.1 as compared to -2.4 +/- 0.1 for the pYEEI p eptide, indicating that in addition to pY, residues conferring peptide bind ing specificity contribute significantly to the salt dependence of SH2 doma in binding This study shows that electrostatic interactions play important roles not only in mediating pY recognition and binding but also in contribu ting to the specificity of the interactions between tyrosyl phosphopeptides and SH2 domains.