DETERMINATION OF THE STRUCTURAL BASIS FOR PICK1 REGULATION

确定 PICK1 监管的结构基础

• 批准号: 8363555
• 负责人: ROBERTO DOMINGUEZ
• 金额: $ 1.19万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363555
• 关键词: Address; Affect; Banana; Binding; C-terminal; Cellular Membrane; Complex; Cytoplasm; Data; Funding; Grant; Learning; Length; Location; Membrane; Memory; N-terminal; National Center for Research Resources; Neurons; Peptides; Phosphotransferases; Positioning Attribute; Principal Investigator; Process; Propionic Acids; Proteins; Radial; Regulation; Relative (related person); Research; Research Infrastructure; Resources; Shapes; Source; Tail; Tertiary Protein Structure; United States National Institutes of Health; amphiphysin; base; cost; env Gene Products; protein structure; receptor; trafficking

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Protein interacting with C-kinase 1 (PICK1) is required for ¿-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor trafficking in neurons, a crucial process involved in learning and memory. PICK1 contains an N-terminal PSD-95/Discs-large/ZO-1 (PDZ) domain followed by a central membrane-binding Bin/amphiphysin/Rvs (BAR) domain and a C-terminal acidic domain. PICK1 is regulated by Ca2+. In the absence of Ca2+, PICK1 is localized to the cytoplasm, suggesting that PICK1 is normally auto-inhibited. Upon Ca2+ stimulation PICK1 binds to the AMPA receptor tail through its PDZ domain and to the adjacent membrane via its BAR domain. Thus, a major conformational change is expected to take place between the Ca2+-bound and Ca2+-free states. We propose to use small-angle x-ray scattering (SAXS) to address how Ca2+ binding affects the structure of PICK1. Specifically, we plan to determine the overall envelope of PICK1 alone and in complex with Ca2+ and an AMPA receptor tail peptide. BAR domains are banana-shaped homodimers that interact with cellular membranes and stabilize or sense membrane curvature. There is a direct correlation between the curvature of the BAR domain and the curvature of the membrane tubules that they stabilize. In the absence of diffracting crystals of the BAR domain of PICK1, we plan to use SAXS to determine the overall shape and radius of curvature of the BAR domain. By comparing the envelop of the BAR domain with that of full length PICK1 will also learn the location of the PDZ domain with respect to the BAR domain. In summary, the SAXS data will provide us a structural understanding of the mechanism of PICK1 activation by Ca2+ and the relative positions of the various domains of PICK1.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 ¿ 需要与 C-激酶 1 (PICK1) 相互作用的蛋白质神经元中的氨基-3-羟基-5-甲基异恶唑-4-丙酸 (AMPA) 受体转运是参与学习和记忆的关键过程，PICK1 包含 N 末端 PSD-95/Discs-large/ZO-1 ( PDZ) 结构域，随后是中央膜结合 Bin/amphiphyn/Rvs (BAR) 结构域和 C 端酸性结构域，由 Ca2+ 调节。在 Ca2+ 缺失的情况下，PICK1 定位于细胞质，这表明 PICK1 在 Ca2+ 刺激下通常是自动抑制的，PICK1 通过其 PDZ 结构域与 AMPA 受体尾部结合，并通过其 BAR 结构域与邻近的膜结合，因此，发生了主要的构象变化。预计发生在 Ca2+ 结合态和 Ca2+ 游离态之间。我们建议使用小角 X 射线散射 (SAXS) 来解决 Ca2+ 结合如何影响具体而言，我们计划确定 PICK1 单独以及与 Ca2+ 和 BAR 受体尾肽复合的整体，它们是与细胞膜相互作用并稳定或感知膜曲率的香蕉形同二聚体。在没有 PICK1 BAR 结构域的衍射晶体的情况下，我们研究了 BAR 结构域的曲率与它们稳定的膜管曲率之间的相关性。计划使用 SAXS 确定 BAR 域的整体形状和曲率半径 通过将 BAR 域的包络线与全长的包络线进行比较，PICK1 还将了解 PDZ 域相对于 BAR 域的位置。 ，SAXS 数据将为我们提供对 Ca2+ 激活 PICK1 的机制以及 PICK1 各个结构域的相对位置的结构理解。