Probing the nature of interactions in SH2 binding interfaces - Evidence from electrospray ionization mass spectrometry

We have adopted nanoflow electrospray ionization mass spectrometry (ESI-MS) and isothermal titration calorimetry (ITC) to probe the mechanism of pepti de recognition by the SH2 domain from the Src family tyrosine kinase protei n, Fyn. This domain is involved in the mediation of intracellular signal tr ansduction pathways by interaction with proteins containing phosphorylated tyrosine (Y*) residues. The binding of tyrosyl phosphopeptides can mimic th ese interactions. Specificity in these interactions has been attributed to the interaction of the Y* and residues proximal and C-terminal to it. Previ ous studies have established that for specific binding with Fyn, the recogn ition sequence consists of pTyr-Glu-Glu-Ile. The specific interactions invo lve the binding of Y* with the ionic, and the Y* + 3 Ile residue with the h ydrophobic binding pockets on the surface of the Fyn SH2 domain. In this wo rk, a variation in the Y* + 3 residue of this high-affinity sequence was ob served to result in changes in the relative binding affinities as determine d in solution (ITC) and in the gas phase (nanoflow ESI-MS). X-ray analysis shows that a feature of the Src family SH2 domains is the involvement of wa ter molecules in the peptide binding site. Under the nanoflow ESI condition s, water molecules appear to be maintained in the Fyn SH2-ligand complex. C ompelling evidence for these molecules being incorporated in the SH2-peptid e interface is provided by the prevalence of the peaks assigned to water-bo und over the water-fret complex at high-energy conditions. Thus, the stabil ity of water protein-ligand complex appears to be intimately Linked to the presence of water.