An intramolecular energetic network regulates ligand recognition in a SH2 domain

In an effort to investigate the molecular determinants of ligand recognition of the C‐terminal SH2 domain of the SHP2 protein, we conducted extensive site‐directed mutagenesis and kinetic binding experiments with a peptide mimicking a specific portion of a physiological ligand (the scaffold protein Gab2). Obtained data provided an in‐depth characterization of the binding reaction, allowing us to pinpoint residues topologically far from the binding pocket of the SH2 domain to have a role in the recognition and binding of the peptide. The presence of a sparse energetic network regulating the interaction with Gab2 was identified and characterized through double mutant cycle analysis, performed by challenging all the designed site‐directed variants of C‐SH2 with a Gab2 peptide mutated at +3 position relative to its phosphorylated tyrosine, a key residue for C‐SH2 binding specificity. Results highlighted non‐optimized residues involved in the energetic network regulating the binding with Gab2, which may be at the basis of the ability of this SH2 domain to interact with different partners in the intracellular environment. Moreover, a detailed analysis of kinetic and thermodynamic parameters revealed the role of the residue at +3 position on Gab2 in the early and late events of the binding reaction with the C‐SH2 domain.