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J. Biol. Chem., Vol. 283, Issue 12, 7338-7345, March 21, 2008

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

Dipak Barua, James R. Faeder¹, and Jason M. Haugh²

From the Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina 27695 and the Department of Computational Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15260

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.

Received for publication, October 9, 2007 , and in revised form, January 18, 2008.

^* This work was supported in part by National Institutes of Health Grant R01-GM067739 and the Cell Migration Consortium under NIGMS, National Institutes of Health Grant U54-GM064346. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.

¹ Supported by National Institutes of Health Grants R37-GM35556 and R01-GM076570 and the Department of Energy through Contract DE-AC52-06NA25396.

² To whom correspondence should be addressed: Box 7905, 911 Partners Way, Raleigh, NC, 27695-7905. Tel.: 919-513-3851; Fax: 919-515-3465; E-mail: jason_haugh{at}ncsu.edu.

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