Abstract

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.

Department(s)

Chemical and Biochemical Engineering

Keywords and Phrases

Amines; Bioactivity; Coordination Reactions; Flow Interactions; Growth (Materials); Platelets; Polypeptides; Proteins; Signal Transduction; Surface Plasmon Resonance; Volumetric Analysis, Alternative Mechanisms; Computational Models; Cooperativity; Do-Mains; Effective Volumes; Growth Factors; Homodimerization; In Vitro; In Vitro Bindings; Intracellular Signals; Isothermal Titration Calorimetries; Kinetic Models; Kinetic Schemes; Multiple Interactions; Orders Of Magnitudes; Phosphoinositide; Receptor Bindings; Receptor Systems; SH2 Domains; Signaling Complexes; SIMPLE Models; Src Homology 2; Structural Constraints; Surface Plasmons, Peptides, Phosphatidylinositol 3 Kinase; Phosphatidylinositol 3 Kinase P85; Platelet Derived Growth Factor Beta Receptor; Protein SH2; Unclassified Drug; Platelet Derived Growth Factor Beta Receptor, Article; Binding Affinity; Binding Competition; Controlled Study; Dimerization; In Vitro Study; Isothermal Titration Calorimetry; Kinetics; Mathematical Model; Priority Journal; Protein Domain; Protein Phosphorylation; Protein Protein Interaction; Protein Structure; Signal Transduction; Surface Plasmon Resonance; Animal; Biological Model; Chemistry; Human; Metabolism; Methodology; Physiology; Src Homology Domain, 1-Phosphatidylinositol 3-Kinase; Animals; Humans; Kinetics; Models, Biological; Receptor, Platelet-Derived Growth Factor Beta; Src Homology Domains; Surface Plasmon Resonance

International Standard Serial Number (ISSN)

0021-9258

Document Type

Article - Journal

Document Version

Final Version

File Type

text

Language(s)

English

Rights

© 2008 American Society for Biochemistry and Molecular Biology, Inc., All rights reserved.

PubMed ID

18204097

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