Calorimetric examination of high-affinity Src SH2 domain-tyrosyl phosphopeptide binding: Dissection of the phosphopeptide sequence specificity and coupling energetics

SH2 domains are protein modules which interact with specific tyrosine phosphorylated sequences in target proteins. The SH2 domain of the Src kinase binds with high affinity to a tyrosine phosphorylated peptide containing the amino acids Glu, Glu, and lie (EEI) at the positions +1, +2, and +3 C-terminal to the phosphotyrosine, respectively. To investigate the degree, of selectivity of the Src SH2 domain for each amino acid of the EEI motif, the binding thermodynamics of a panel of substitutions at the +1 (Gln, Asp, Ala, Cry), +2 (Gln, Asp, Ala, Gly:), and +3 (Leu, Val, Ala, Gly) positions were examined using titration microcalorimetry. It was revealed that the Src SH2 domain is insensitive (Delta Delta G degrees less than or equal to 0.6 kcal/mol) to conservative substitutions at all three peptide positions. However, mutation to Ala resulted in moderate reductions in Delta G degrees, with the substitution at the +3 position showing the largest loss in affinity (Delta Delta G degrees = 1.4 kcal/mol), followed by the +2 (Delta Delta G degrees = 1.0 kcal/mol) and +1 (Delta Delta G degrees = 0.5 kcal/mol) positions. This hierarchy of binding was not reflected in the values of the heat capacity change, since only the peptide substituted to Ala at the +3 position showed a Delta C(p)degrees that was reduced in magnitude compared to wild-type. To assess the degree of cooperation upon binding (or coupling) between the amino acids of the EEI sequence, the binding of a series of singly, doubly, and triply Ala substituted phosphopeptides was examined and analyzed using double mutant cycles. It was revealed that the effects of the Ala substitutions on Delta G degrees were additive. However, nonadditive binding enthalpies were observed between the +1 Glu and +3 Ile, as well as the +2 Glu and +3 Ile, suggesting that communication occurs between residues of the EEI motif upon binding.