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

The binding of tyrosine phosphorylated targets by SH2 domains is required f or propagation of many cellular signals in higher eukaryotes; however, the determinants of phosphotyrosine (pTyr) recognition by SH2 domains are not w ell understood. In order to identify the attributes of pTyr required for hi gh affinity interaction with SH2 domains, the binding of the SH2 domain of the Src kinase (Src SH2 domain) to a dephosphorylated peptide, a phosphoser ine-containing peptide, and the amino acid pTyr was studied using titration calorimetry and compared with the binding of a high affinity tyrosyl phosp hopeptide. The dephosphorylated peptide and the phosphoserine containing pe ptide both bind extremely weakly to the Src SH2 domain (Delta G degrees (de phosphorylated) = -3.6 kcal/mol, Delta G degrees (phosphoserine) > -3.7 kca l/mol); however, the Delta G degrees value of pTyr binding is more favorabl e (-4.7 kcal/mol, or 50 % of the entire binding free energy of a high affin ity tyrosyl phosphopeptide). These results indicate that both the phosphate and the tyrosine ring of the pTyr are critical determinants of high affini ty binding. Alanine mutagenesis was also used to evaluate the energetic con tribution to binding of ten residues located in the pTyr-binding site. Muta tion of the strictly conserved Arg beta B5 resulted in a large increase in Delta G degrees (Delta Delta G degrees = 3.2 kcal/mol) while elimination of the other examined residues each resulted in a significantly smaller (Delt a Delta G degrees < 1.4 kcal/mol) reduction in affinity, indicating that Ar g beta B5 is the single most important determinant of pTyr recognition. How ever, mutation of Cys beta C3, a residue unique to the Src SH2 domain, surp risingly increased affinity by eightfold (Delta Delta G degrees = - 1.1 kca l/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-termi nal to the pTyr. In addition, comparison of each residue's Delta Delta G de grees value upon mutation with that residue's sequence conservation among S H2 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 cr itical interaction, the buried ionic bond between the phosphate of the pTyr and Arg beta B5 of the SH2 domain, driving the binding of SH2 domains to t yrosine phosphorylated targets. (C) 1999 Academic Press.