PP1:NIPP1 HOLOENZYME

PP1:NIPP1全酶

• 批准号: 8363375
• 负责人: Rebecca Page
• 金额: $ 0.4万
• 依托单位: BROOKHAVEN SCIENCE ASSOC-BROOKHAVEN LAB
• 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/8363375
• 关键词: Active Sites; Binding; Binding Sites; Catalytic Domain; Cell Cycle Regulation; Cell division; Cell physiology; Complex; Crystallization; Crystallography; DNA Repair; Data; Enzymes; Eukaryota; Event; Funding; Gene Silencing; Grant; Holoenzymes; Light; Molecular Conformation; National Center for Research Resources; Needles; Nuclear; Nuclear Protein; Principal Investigator; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; RNA Splicing; Regulation; Research; Research Infrastructure; Resources; Sampling; Site; Source; Surface; Synchrotrons; Toxin; United States National Institutes of Health; Work; cost; inhibitor/antagonist; insight; interest; prevent; research study; small molecule; two-dimensional

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. Phosphoprotein Phosphatase 1, PP1, is a ubiquitous serine/threonine phosphatase (330 residues, ~ 38 kDa) expressed in all eukaryotes that regulates a wide array of cellular processes. The promiscuity of PP1 is greatly reduced by interaction with several inhibitory and targeting proteins. By interacting with residues of the active site of PP1, inhibitory proteins prevent to access catalytic residues of the enzyme. The targeting proteins relocate PP1 to a particular cellular milieu and interact with the surface of the catalytic core in such a way that only certain binding sites are exposed, whereas, others are sites are sterically occluded. Current crystallographic studies of PP1 in complex with small molecule toxins, inhibitory proteins and targeting proteins have revealed that the surface and conformation of the catalytic core remains invariant; however, all of the proteins studied to date have shown markeedly different themes in their interactions with the PP1. To shed new insights on PP1 regulation by targeting proteins, we have initiated work with one such protein, NIPP (Nuclear Inhibitor of PP1). NIPP targets PP1 after interaction with various proteins implicated in four different cellular processes (cell division, gene silencing, DNA repair and RNA splicing). Since targeting of NIPP1 to PP1 is a major event in cell cycle regulation; thus, we are interested in understanding this relationship at the atomic level. After exhaustive biophysical characterization of the monomeric form of the PP1-binding domain of NIPP, we have moved to studying the holoenzyme complex; to this end, we have determined the Kd with ITC (low nM), used data from complex NMR experiments to optimize samples for crystallization trails and attained two dimensional needle-like crystals.

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。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。 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。 磷蛋白磷酸酶 1 (PP1) 是一种普遍存在的丝氨酸/苏氨酸磷酸酶（330 个残基，~ 38 kDa），在所有真核生物中表达，调节多种细胞过程。 PP1 的混杂性通过与几种抑制蛋白和靶向蛋白的相互作用而大大减少。 通过与 PP1 活性位点的残基相互作用，抑制蛋白阻止接触酶的催化残基。 靶向蛋白将 PP1 重新定位到特定的细胞环境，并与催化核心表面相互作用，从而仅暴露某些结合位点，而其他位点则被空间封闭。 目前PP1与小分子毒素、抑制蛋白和靶向蛋白复合物的晶体学研究表明，催化核心的表面和构象保持不变；然而，迄今为止研究的所有蛋白质在与 PP1 的相互作用中都表现出明显不同的主题。 为了通过靶向蛋白质来获得关于 PP1 调节的新见解，我们开始研究这样一种蛋白质：NIPP（PP1 核抑制剂）。 NIPP 在与四种不同细胞过程（细胞分裂、基因沉默、DNA 修复和 RNA 剪接）中涉及的各种蛋白质相互作用后，以 PP1 为目标。 由于 NIPP1 靶向 PP1 是细胞周期调节中的一个重大事件；因此，我们有兴趣在原子层面理解这种关系。 在对 NIPP PP1 结合域的单体形式进行详尽的生物物理表征后，我们开始研究全酶复合物；为此，我们用 ITC（低 nM）确定了 Kd，使用复杂 NMR 实验的数据来优化样品的结晶轨迹，并获得了二维针状晶体。