Alternative modes of binding of proteins with tandem SH2 domains

The issue of specificity in tyrosine kinase intracellular signaling mediate d by src homology 2 (SH2) domains has great importance in the understanding how individual signals maintain their mutual exclusivity and affect downst ream responses. Several proteins contain tandem SH2 domains that, on intera cting with their ligand, provide a higher level of specificity than can be afforded by the interaction of a single SH2 domain. In this study, we focus on the comparison of two proteins ZAP70 and the p85 subunit of PI 3-kinase , which although distinctly different in function and general structure, po ssess tandem SH2 domains separated by a Linker region and which bind to pho sphorylated receptor molecules localized to the cell membrane. Binding stud ies using isothermal titration calorimetry show that these two proteins int eract with peptides mimicking their physiological ligands in very different ways. In the case of the SH2 domains from ZAP70, they interact with a stoi chiometry of unity, while p85 is able to make two distinct interactions, on e with a stoichiometry of 1:1 and the other with two p85 molecules interact ing with one receptor. The observation of two different modes of binding of p85 might be important in providing different cellular responses based on fluctuating intracellular concentration regimes of this protein. Thermodyna mic data on both proteins suggest that a conformational change occurs on bi nding. On investigation of this structural change using a truncated form of p85 (including just the two SH2 domains and the inter-SH2 region), both NM R and circular dichroism spectroscopic studies failed to show significant c hanges in secondary structure. This suggests that any conformational change associated with binding is small and potentially limited to loop regions o f the protein.