Computational Models of Tandem Src Homology 2 Domain Interactions and Application to Phosphoinositide 3-Kinase*

Intracellular signal transduction proteins typically utilize multiple interaction domains for proper targeting, and thus a broad diversity of distinct signaling complexes may be assembled. Considering the coordination of only two such domains, as in tandem Src homology 2 (SH2) domain constructs, gives rise to a kinetic scheme that is not adequately described by simple models used routinely to interpret in vitro binding measurements. To analyze the interactions between tandem SH2 domains and bisphosphorylated peptides, we formulated detailed kinetic models and applied them to the phosphoinositide 3-kinase p85 regulatory subunit/platelet-derived growth factor β-receptor system. Data for this system from different in vitro assay platforms, including surface plasmon resonance, competition binding, and isothermal titration calorimetry, were reconciled to estimate the magnitude of the cooperativity characterizing the sequential binding of the high and low affinity SH2 domains (C-SH2 and N-SH2, respectively). Compared with values based on an effective volume approximation, the estimated cooperativity is 3 orders of magnitude lower, indicative of significant structural constraints. Homodimerization of full-length p85 was found to be an alternative mechanism for high avidity binding to phosphorylated platelet-derived growth factor receptors, which would render the N-SH2 domain dispensable for receptor binding.