Investigation of phosphotyrosine recognition by the SH2 domain of the Src kinase

Abstract

The binding of tyrosine phosphorylated targets by SH2 domains is required for propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not well understood. In order to identify the attributes of pTyr required for high affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoserine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosphopeptide. The dephosphorylated peptide and the phosphoserine containing peptide both bind extremely weakly to the Src SH2 domain (ΔGo (dephosphorylated) = −3.6 kcal/mol, ΔGo (phosphoserine) > −3.7 kcal/mol); however, the ΔGo value of pTyr binding is more favorable (−4.7 kcal/mol, or 50% of the entire binding free energy of a high affinity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affinity binding. Alanine mutagenesis was also used to evaluate the energetic contribution to binding of ten residues located in the pTyr-binding site. Mutation of the strictly conserved Arg βB5 resulted in a large increase in ΔGo (ΔΔGo = 3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (ΔΔGo < 1.4 kcal/mol) reduction in affinity, indicating that Arg βB5 is the single most important determinant of pTyr recognition. However, mutation of Cys βC3, a residue unique to the Src SH2 domain, surprisingly increased affinity by eightfold (ΔΔGo = −1.1 kcal/mol). Using a double mutant cycle analysis, it was revealed that residues of the pTyr-binding pocket are not coupled to the peptide residues C-terminal to the pTyr. In addition, comparison of each residue’s ΔΔGo value upon mutation with that residue’s sequence conservation among SH2 domains revealed only a modest correlation between a residue’s energetic contribution to pTyr recognition and its conservation throughout evolution. The results of this investigation highlight the importance of a single critical interaction, the buried ionic bond between the phosphate of the pTyr and Arg βB5 of the SH2 domain, driving the binding of SH2 domains to tyrosine phosphorylated targets.