title: Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer.
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 concentration-dependent 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.
authors.affiliation: From the Department of Molecular Pharmacology.
authors.forename: Jaclyn
authors.initials: J
authors.lastname: LoPiccolo
authors.affiliation: the Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94158, and.
authors.forename: Seung Joong
authors.initials: SJ
authors.lastname: Kim
authors.affiliation: the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065.
authors.forename: Yi
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Bin
authors.initials: B
authors.lastname: Wu
authors.affiliation: From the Department of Molecular Pharmacology.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Wu
authors.affiliation: the Laboratory of Mass Spectrometry and Gaseous Ion Chemistry, The Rockefeller University, New York, New York 10065.
authors.forename: Brian T
authors.initials: BT
authors.lastname: Chait
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Singer
authors.affiliation: the Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, and California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, California 94158, and.
authors.forename: Andrej
authors.initials: A
authors.lastname: Sali
authors.affiliation: Department of Biochemistry, michael.brenowitz@einstein.yu.edu.
authors.forename: Michael
authors.initials: M
authors.lastname: Brenowitz
authors.affiliation: Department of Biochemistry, anne.bresnick@einstein.yu.edu.
authors.forename: Anne R
authors.initials: AR
authors.lastname: Bresnick
authors.affiliation: From the Department of Molecular Pharmacology, Department of Biochemistry, jonathan.backer@einstein.yu.edu.
authors.forename: Jonathan M
authors.initials: JM
authors.lastname: Backer
mesh_terms: D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
chemical_list: D058543:Class Ia Phosphatidylinositol 3-Kinase
keywords: analytical ultracentrifugation; molecular modeling; phosphatidylinositide 3-kinase (PI 3-kinase); phosphatidylinositol signaling; small-angle X-ray scattering (SAXS); structural model