Structures and mechanisms of nuclear import and export

核进出口的结构和机制

• 批准号: 7344817
• 负责人: Yuh Min Chook
• 金额: $ 26.09万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7344817
• 关键词: Address; Binding; Biochemical; Cell Cycle Progression; Cell Nucleus; Cell physiology; Cellular Apoptosis Susceptibility Protein; Complex; Cytoplasm; Data; Dissociation; Family; Gene Expression; Goals; Guanosine Triphosphate Phosphohydrolases; Karyopherins; Knowledge; Ligands; Mediating; Modeling; Molecular; Molecular Structure; Nuclear Export; Nuclear Import; Nuclear Localization Signal; Pathway interactions; Peptide Signal Sequences; Process; Proteins; Research; Research Personnel; Running; Signal Transduction; Specificity; Structure; Time; Work; X-Ray Crystallography; base; comparative; hnRNP A1; insight; nucleocytoplasmic transport; programs; ran-binding protein 1

DESCRIPTION (provided by applicant): Proteins in the Karyopherinbeta (Kapbeta) family mediate multiple macromolecular import and export pathways between the nucleus and the cytoplasm. The binding of transport substrates and the GTPase Ran to import- Kapbetas is mutually exclusive, while the binding of substrates and Ran to export-Kapbetas is cooperative. This difference in cooperativity is the main biochemical distinction between nuclear import and export, and is likely the key factor in determining nuclear transport directions. A major question in the field concerns mechanistic distinctions between nuclear import and export. It is not known how the homologous import and export-Kapbeta proteins have such disparities in Ran binding activities, cooperativities in Ran and substrate binding, and how they arrive at opposing directions of transport. To address these questions, this proposal describes comparative structural analyses by X-ray crystallography of interactions between import factor Kapbeta2 and export factor CAS with their ligands. The goal of our first aim is to determine the crystal structures of Kaplbeta2 bound to nuclear localization signals (NLSs) of import substrates hnRNP A1 and TAP. These NLSs are non-homologous and are also classified as non-classical NLSs. Structures of their complexes will reveal specificity determinants for Kapbeta2, and inform on the mechanism of substrate/non-classical NLS recognition. Our second aim is to determine the structure of free Kapbeta2. Import by Kapbeta2 begins in the cytoplasm with the unliganded karyopherin. Thus, understanding of the whole import pathway and changes in structure induced by each ligand will require structural studies of the free state as well as of all the complexes. The third and fourth aims will focus on nuclear export. We will determine the crystal structure of the CAS-Kapalpha-RanGppNHp complex. This work will explain for the first time how an export substrate is recognized, it will also will explain the structural basis of positive cooperativity in Ran and export substrate binding by an export-Kapbeta. Finally, we will solve the structure of the Cas-RanGppNHp-RanBP1 complex, a crucial intermediate in the export dissociation process. This work will allow us to understand how export substrate, Kapalpha, is dissociated in the cytoplasm in the last steps of nuclear export. Collectively, these studies will provide insights into specific mechanisms of the different steps in nuclear import by Kapbeta2 and nuclear export by CAS. But more importantly, comparison between these structures of import and export complexes will reveal the mechanistic distinctions between nuclear import and export.

描述（由申请人提供）：Karyopherinbeta（Kap beta）家族中的蛋白质介导细胞核和细胞质之间的多种大分子输入和输出途径。转运底物和GTTRan Ran与输入-Kappetas的结合是相互排斥的，而底物和Ran与输出-Kappetas的结合是合作的。这种协同性的差异是核输入和输出之间的主要生化区别，并且可能是决定核运输方向的关键因素。这一领域的一个主要问题涉及核进口和核出口之间的机械区别。尚不清楚同源的输入和输出-Kap β蛋白如何在Ran结合活性、Ran和底物结合的协同性以及它们如何到达相反的运输方向中具有这样的差异。为了解决这些问题，该提案描述了比较结构分析的X射线晶体学的输入因子Kapbeta 2和输出因子CAS与它们的配体之间的相互作用。我们的第一个目标是确定与输入底物hnRNPA 1和TAP的核定位信号（NLS）结合的Kap 1 β 2的晶体结构。这些NLS是非同源的，也被归类为非经典NLS。其复合物的结构将揭示Kapbeta 2的特异性决定簇，并告知底物/非经典NLS识别的机制。我们的第二个目标是确定自由Kapbeta 2的结构。通过Kapbeta 2的输入开始于细胞质中的未配体的核转运蛋白。因此，了解整个进口途径和结构变化引起的每个配体将需要结构研究的自由状态以及所有的配合物。第三和第四个目标将集中在核出口方面。我们将确定CAS-Kapalpha-RanGppNHp复合物的晶体结构。这项工作将首次解释输出底物是如何被识别的，它也将解释Ran和输出底物通过输出-Kappa结合的正协同性的结构基础。最后，我们将解决Cas-RanGppNHp-RanBP 1复合物的结构，这是输出解离过程中的关键中间体。这项工作将使我们能够了解输出底物Kapalpha在核输出的最后步骤中如何在细胞质中解离。总的来说，这些研究将提供深入了解的具体机制的不同步骤的核进口的Kapbeta 2和核出口的CAS。但更重要的是，这些进口和出口复合体结构之间的比较将揭示核进口和核出口之间的机械区别。