A thermodynamic analysis using isothermal titration calorimetry (ITC) has been performed to examine the binding interaction between the SH2 (Src homology 2) domain of growth factor receptor binding protein 2 (Grb2-SH2) and one of its phosphotyrosine (pY) polypeptide ligands. Interaction of the Shc-derived phosphotyrosine hexapeptide Ac-SpYVNVQ-NH2 with Grb2-SH2 was both enthalpically and entropically favorable (DeltaH = -7.55 kcal mol-1, -TDeltaS = -1.46 kcal mol-1 , DeltaG = -9.01 kcal mol-1, T = 20 degrees C). ITC experiments using five alanine-substituted peptides were performed to examine the role of each side chain in binding. The results were consistent with homology models of the Grb2-SH2-Shc hexapeptide complex which identified several possible hydrogen bonds between Grb2-SH2 and the phosphotyrosine and conserved asparagine(+2) side chains of the Shc hexapeptide. These studies also demonstrated that the hydrophobic valine(+1) side chain contributes significantly to the favorable entropic component of binding. The thermodynamic and structural data are consistent with a Grb2-SH2 recognition motif of pY-hydrophobic-N-X (where X is any amino acid residue). The measured heat capacity of binding (DeltaCp = -146 cal mol-1 K-1) was very similar to computed values using semiempirical estimates (DeltaCp = -106 to -193 cal mol-1 K-1) derived from apolar and polar accessible surface area values calculated from several homology models of the Grb2-SH2-Shc hexapeptide complex. The homology model which most closely reproduced the measured DeltaCp value is also the model which had the lowest RMS deviation from the subsequently determined crystal structure. Calculations based on the thermodynamic data and these semiempirical estimates indicated that the binding event involves burial of nearly comparable apolar (677 A2) and polar (609 A2) surface areas.