Assembly and molecular architecture of the phosphoinositide 3-kinase p85α homodimer

Jaclyn LoPiccolo, Seung Joong Kim, Yi Shi, Bin Wu, Haiyan Wu, Brian T. Chait, Robert H. Singer, Andrej Sali, Michael D. Brenowitz, Anne R. Bresnick, Jonathan M. Backer

Research output: Contribution to journalArticle

10 Scopus citations

Abstract

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization. Our in vitro assembly and structural analyses have been enabled by the creation of cysteine-free p85α that is functionally equivalent to native p85α. Analytical ultracentrifugation studies showed that p85α undergoes rapidly reversible monomer-dimer assembly that is highly exothermic in nature. In addition to the documented SH3-PR1 dimerization interaction, we identified a second intermolecular interaction mediated by cSH2 domains at the C-terminal end of the polypeptide. We have demonstrated in vivo concentrationdependent dimerization of p85α using fluorescence fluctuation spectroscopy. Finally, we have defined solution conditions under which the protein is predominantly monomeric or dimeric, providing the basis for small angle x-ray scattering and chemical cross-linking structural analysis of the discrete dimer. These experimental data have been used for the integrative structure determination of the p85α dimer. Our study provides new insight into the structure and assembly of the p85α homodimer and suggests that this protein is a highly dynamic molecule whose conformational flexibility allows it to transiently associate with multiple binding proteins.

Original languageEnglish (US)
Pages (from-to)30390-30405
Number of pages16
JournalJournal of Biological Chemistry
Volume290
Issue number51
DOIs
StatePublished - Dec 18 2015

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

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