Structures and mechanisms of nuclear import and export

描述（由申请人提供）：核紫蛋白（Kapbeta）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。转运底物与GTPase Ran结合进口- kapbeta是相互排斥的，而底物与Ran结合出口- kapbeta是合作的。这种协同性的差异是核进口和核出口之间的主要生化区别，可能是决定核运输方向的关键因素。该领域的一个主要问题涉及核进出口之间的机械区别。目前尚不清楚同源输入和输出的κ β蛋白在Ran结合活性、Ran和底物结合的合作性以及它们如何到达相反的运输方向方面是如何存在差异的。为了解决这些问题，本提案描述了进口因子Kapbeta2和出口因子CAS与其配体之间相互作用的x射线晶体学比较结构分析。我们的第一个目标是确定Kaplbeta2与进口底物hnRNP A1和TAP的核定位信号（NLSs）结合的晶体结构。这些NLSs是非同源的，也被归类为非经典NLSs。它们复合物的结构将揭示Kapbeta2的特异性决定因素，并为底物/非经典NLS识别的机制提供信息。我们的第二个目标是确定游离Kapbeta2的结构。Kapbeta2的输入开始于细胞质中的无配体核细胞蛋白。因此，了解整个进口途径和每个配体引起的结构变化将需要对自由状态以及所有配合物进行结构研究。第三和第四个目标将侧重于核出口。我们将确定CAS-Kapalpha-RanGppNHp配合物的晶体结构。这项工作将首次解释出口底物是如何被识别的，它也将解释Ran和出口底物结合的正向合作的结构基础。最后，我们将解决Cas-RanGppNHp-RanBP1复合物的结构，这是出口解离过程中的关键中间体。这项工作将使我们了解输出底物Kapalpha在核输出的最后步骤中是如何在细胞质中解离的。总的来说，这些研究将为Kapbeta2的核进口和CAS的核出口的不同步骤的具体机制提供见解。但更重要的是，对这些进出口综合体结构的比较将揭示核进出口之间的机制区别。

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.