Thermodynamic measurements, structural determinations,and molecular computations wereapplied to a series of peptide ligands of the pp60
c-srcSH2 domain in an attempt to understand the criticalbinding determinants for this class of molecules. Isothermaltitration calorimetry (ITC) measurementswere combined with structural data derived from X-ray crystallographicstudies on 12 peptide-SH2 domaincomplexes. The peptide ligands studied fall into two generalclasses: (1) dipeptides of the generalframework
N-acetylphosphotyrosine (or phosphotyrosinereplacement)-Glu or methionine (or S-methylcysteine)-X, where X represents a hydrophobic amine, and (2) tetra- orpentapeptides of the generalframework
N-acetylphosphotyrosine-Glu-Glu-Ile-X, where Xrepresents either Glu, Gln, or NH
2.Dipeptideanalogs which featured X as either hexanolamine or heptanolamine wereable to pick up new hydrogenbonds involving their hydroxyl groups within a predominantly lipophilicsurface cavity. However, dueto internal strain as well as the solvent accessibility of the newhydrogen bonds formed, no net increasein binding affinity was observed. Phosphatase-resistantbenzylmalonate and
,
-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity forthe pp60
c-src SH2 domain butcaused local structural perturbations in the phosphotyrosine-bindingsite. In the case where a reversiblecovalent thiohemiacetal was formed between a formylated phosphotyrosineanalog and the thiol side chainof Cys-188,
S was 25.6 cal/(mol K) lower than for thenonformylated phosphotyrosine parent. Normalmode calculations show that the dramatic decrease in entropy observedfor the covalent thiohemiacetalcomplex is due to the inability of the phosphotyrosine moiety totransform lost rotational and translationaldegrees of freedom into new vibrational modes.