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.