Structural Basis for RCC1 Directed Recruitment of Ran GTPase to Chromatin

描述（由申请人提供）：真核细胞必须能够在其细胞核和细胞质之间定向运输大分子，并通过有丝分裂分裂细胞。这些基本过程是通过将小的Ran鸟苷三磷酸酶（GTTP）蛋白定位在细胞质或细胞核内的核苷酸结合状态，以及通过在染色体周围产生RanGTP的梯度来控制的。RanGTP在细胞核中的这种空间定位是通过染色质结合的RCC 1（染色体凝聚调节因子）蛋白实现的。RCC 1将Ran招募到染色体的核小体重复单位，并促进RanGDP与RanGTP的交换，从而在染色体周围产生高浓度的RanGTP。因此，Ran/RCC 1/核小体复合物调节真核细胞正常发挥功能的关键基础过程。 我们的生化研究和在上一个资助期确定的RCC 1/核小体的晶体结构显示了RCC 1如何与核小体结合。这些研究也提出了一些新的问题，包括（a）Ran/RCC 1/核小体复合物中Ran的哪些区域与RCC 1/核小体相互作用，（B）Ran结合后RCC 1/核小体构象变化的作用以及（c）Ran和RCC 1如何与核小体相互作用的可能物种差异。因此，我们的目标是描述如何RCC 1和RAN从不同的物种相互作用的核小体核心粒子的原子细节。 这项建议着重于三个具体目标：1.确定Ran/RCC 1/核小体复合物的结构。我们将使用结晶前和结晶后策略来改进Ran/RCC 1/核小体复合物的单晶，以确定Ran/RCC 1/核小体复合物的原子结构。2.定义Ran/RCC 1/核小体复合物中Ran如何与核小体相互作用。我们提出了生化研究，以了解哪些Ran区域与来自不同物种的Ran/RCC 1/核小体复合物中的核小体相互作用，以及这些相互作用如何影响Ran的核苷酸交换活性。3.确定构象变化在RCC 1/核小体和Ran/RCC 1/核小体复合物中的作用。我们将使用安装在核小体上的荧光探针来研究Ran和RCC 1如何与核小体相互作用，以及构象变化在这种结合中的作用。

DESCRIPTION (provided by applicant): A eukaryotic cell must be able to transport macromolecules directionally between its nucleus and cytoplasm, and to divide the cell through mitosis. These fundamental processes are controlled by localizing the small Ran guanosine triphosphatase (GTPase) protein in its nucleotide bound state within the cytoplasm or the nucleus, and by generating a gradient of RanGTP around the chromosomes. This spatial localization of RanGTP in the nucleus is achieved through chromatin bound RCC1 (regulator of chromosome condensation) protein. RCC1 recruits Ran to the nucleosome repeating unit of chromosomes and promotes the exchange of RanGDP for RanGTP, thereby creating a high concentration of RanGTP around chromosomes. The Ran/RCC1/nucleosome complex thus regulates fundamental processes critical for a eukaryotic cell to function properly. Our biochemical studies and crystal structure of the RCC1/nucleosome determined in the last funding period showed how RCC1 binds to the nucleosome. These studies also present new questions about the Ran/RCC1/nucleosome complex, including (a) what regions of Ran interact with RCC1/nucleosome in the Ran/RCC1/nucleosome complex, (b) the role of conformational changes in RCC1/nucleosome upon Ran binding and (c) possible species differences in how Ran and RCC1 interact with the nucleosome. Our goal is therefore to describe how RCC1 and Ran from different species interact with the nucleosome core particle in atomic detail. This proposal focuses on three specific aims: 1. Determine the structure of the Ran/RCC1/nucleosome complex. We will improve single crystals of the Ran/RCC1/nucleosome complex using pre- and post-crystallization strategies to determine the atomic structure of the Ran/RCC1/nucleosome complex. 2. Define how Ran interacts with the nucleosome in Ran/RCC1/nucleosome complexes. We propose biochemical studies to understand which Ran regions interact with the nucleosome in Ran/RCC1/nucleosome complexes from different species, and how these interactions affect Ran's nucleotide exchange activity. 3. Determine role of conformational changes in RCC1/nucleosome & Ran/RCC1/nucleosome complexes. We will use fluorescent probes installed on the nucleosome to study how Ran and RCC1 interact with the nucleosome, and the role of conformational changes in this binding.