J.M. Bradshaw, R.A. Grucza, J.E. Ladbury, G. Waksman.
Probing the "two pronged-plug two holed-socket" model for the mechanism of binding of the Src SH2 domain to phosphotyrosyl peptides: a thermodynamic study. Biochemistry. In Press.

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SH2 domains are proteins modules which specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two pronged-plug two holed-socket" mechanism whereby binding is hypothesized to resemble a two pronged-plug (the peptide) inserting into a two holed-socket (the SH2 domain). This binding model predicts 1) a hydrophobic basis for high affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and 2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (DeltaGo, DeltaHo, DeltaSo, DeltaCpo) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides which have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism which does not involve substantial conformational changes, the DeltaCpo for all peptides were small and, at least for the high affinity interactions, similar to the DeltaCpo predicted from surface area calculations. However, unexpectedly, this study reveals that high affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the DeltaCpo values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two pronged-plug two holed-socket" model is an oversimplification of the Src SH2 domain binding mechanism.

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