DETERMINATION OF THE STRUCTURAL BASIS FOR PICK1 REGULATION

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 与C-激酶1(PICK1)相互作用的蛋白是¿-amino-3-hydroxy-5-methylisoxazole-4-propionic酸(AMPA)受体在神经元中运输所必需的，这是一个涉及学习和记忆的关键过程。PICK1含有一个N-末端的PSD-95/Discs-Large/ZO-1(PDZ)结构域，接着是一个中央的膜结合Bin/两面体蛋白/RVS(BAR)结构域和一个C-末端的酸性结构域。PICK1受钙离子调控。在没有钙的情况下，PICK1定位于细胞质，这表明PICK1通常是自动抑制的。在钙离子刺激下，PICK1通过其PDZ结构域与AMPA受体尾部结合，并通过其BAR结构域与相邻膜结合。因此，钙结合状态和无钙状态之间有望发生重大的构象变化。我们建议使用小角X射线散射(SAXS)来研究钙结合如何影响PICK1的结构。具体地说，我们计划确定PICK1单独以及与钙离子和AMPA受体尾肽的复合体的总包膜。杆状结构域是香蕉形状的同源二聚体，它与细胞膜相互作用，稳定或感觉细胞膜的曲率。在杆域的曲率和它们稳定的膜小管的曲率之间存在着直接的关联。在没有PICK1棒区的衍射晶体的情况下，我们计划用SAXS来确定棒区的整体形状和曲率半径。通过将BAR结构域的包络与全长PICK1的包络进行比较，PICK1还将了解PDZ结构域相对于BAR结构域的位置。综上所述，SAXS数据将使我们对PICK1被钙激活的机制以及PICK1各个结构域的相对位置有一个结构性的了解。

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.