PEPTIDE LIGANDS OF PP60(C-SRC) SH2 DOMAINS - A THERMODYNAMIC AND STRUCTURAL STUDY

Thermodynamic measurements, structural determinations, and molecular c omputations were applied to a series of peptide ligands of the pp60(c- src) SH2 domain in an attempt to understand the critical binding deter minants for this class of molecules. Isothermal titration calorimetry (ITC) measurements were combined with structural data derived from X-r ay crystallographic studies on 12 peptide-SH2 domain complexes. The pe ptide ligands studied fall into two general classes: (1) dipeptides of the general framework N-acetylphosphotyrosine (or phosphotyrosine rep lacement)-Glu or methionine (or S-methyl-cysteine)-X, where X represen ts a hydrophobic amine, and (2) tetra- or pentapeptides of the general framework N-acetylphosphotyrosine-Glu-Glu-Ile-X, where X represents e ither Glu, Gln, or NH2. Dipeptide analogs which featured X as either h exanolamine or heptanolamine were able to pick up new hydrogen bonds i nvolving their hydroxyl groups within a predominantly lipophilic surfa ce cavity. However, due to internal strain as well as the solvent acce ssibility of the new hydrogen bonds formed, no net increase in binding affinity was observed. Phosphatase-resistant benzylmalonate and alpha ,alpha-difluorobenzyl phosphonate analogs of phosphotyrosine retained some binding affinity for the pp60(c-src) SH2 domain but caused local structural perturbations in the phosphotyrosine-binding site. In the c ase where a reversible covalent thiohemiacetal was formed between a fo rmylated phosphotyrosine analog and the thiol side chain of Cys-188, D elta S was 25.6 cal/(mol K) lower than for the nonformylated phosphoty rosine parent. Normal mode calculations show that the dramatic decreas e in entropy observed for the covalent thiohemiacetal complex is due t o the inability of the phosphotyrosine moiety to transform lost rotati onal and translational degrees of freedom into new vibrational modes.