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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 磷酸蛋白磷酸酶1，PP1是无处不在的丝氨酸/苏氨酸磷酸酶（330个残基，〜38 kDa），在调节广泛的细胞过程的所有真核生物中表达。 通过与几种抑制性和靶向蛋白的相互作用，PP1的滥交大大降低了。 通过与PP1活性位点的残基相互作用，抑制性蛋白会阻止访问酶的催化残基。 靶向蛋白将PP1重新定位为特定的细胞环境，并与催化核的表面相互作用，使得只有某些结合位点暴露了，而其他位置则在空间上被遮住。 PP1在与小分子毒素，抑制蛋白和靶向蛋白的复合物中的当前晶体学研究表明，催化核的表面和构象仍然不变。但是，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了越来越不同的主题。 为了通过靶向蛋白质对PP1调节进行新的见解，我们已经与一种这样的蛋白质NIPP（PP1的核抑制剂）开始了工作。 NIPP与与四种不同的细胞过程相互作用（细胞分裂，基因沉默，DNA修复和RNA剪接）相互作用后的PP1。 由于将NIPP1靶向PP1是细胞周期调节中的重大事件。因此，我们有兴趣在原子层面理解这种关系。 在NIPP的PP1结合结构域的单体形式的详尽生物物理表征之后，我们已转向研究全酶复合物。为此，我们已经用ITC（低NM）确定了KD，该数据使用了来自复杂NMR实验的数据，以优化样品以进行结晶径，并获得了两个尺寸的针状晶体。