LoPiccolo Jaclyn, Kim Seung Joong, Shi Yi, Wu Bin, Wu Haiyan, Chait Brian T, Singer Robert H, Sali Andrej, Brenowitz Michael, Bresnick Anne R, Backer Jonathan M
From the Department of Molecular Pharmacology.
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.
J Biol Chem. 2015 Dec 18;290(51):30390-405. doi: 10.1074/jbc.M115.689604. Epub 2015 Oct 16.
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.
磷脂酰肌醇3-激酶（PI3K）是一类脂质激酶家族，可被生长因子和G蛋白偶联受体激活，并传递细胞内信号以促进生长、存活、增殖和代谢。p85α是一种由五个结构域组成的模块化蛋白，它结合并抑制IA类PI3K催化亚基的酶活性。在此，我们基于体内和体外溶液表征数据描述了p85α二聚体的结构状态。通过创建功能上等同于天然p85α的无半胱氨酸p85α，我们实现了体外组装和结构分析。分析超速离心研究表明，p85α经历快速可逆的单体-二聚体组装，本质上是高度放热的。除了已记录的SH3-PR1二聚化相互作用外，我们还确定了多肽C末端由cSH2结构域介导的第二种分子间相互作用。我们使用荧光涨落光谱法在体内证明了p85α的浓度依赖性二聚化。最后，我们确定了蛋白质主要为单体或二聚体的溶液条件，为离散二聚体的小角X射线散射和化学交联结构分析提供了基础。这些实验数据已用于p85α二聚体的整合结构测定。我们的研究为p85α同型二聚体的结构和组装提供了新的见解，并表明该蛋白是一种高度动态的分子，其构象灵活性使其能够与多种结合蛋白瞬时结合。
