The dynamic mechanism of nuclear transport visualized at the atomic scale

大分子核质转运的唯一介质是核孔复合物（NPC）， 由称为核孔蛋白（Nups）的蛋白质组成。这些核孔蛋白 假设核转运因子（NTR）相关货物的选择性扩散涉及相对 转运因子和富含苯丙氨酰甘氨酰（FG）的重复区域之间的弱相互作用， Nups（FG-Nups）提供选择性，同时连续高度的FG-重复区动态无序 为工艺提供足够的速度，有助于选择性扩散。核输运缺陷与NPC 与许多疾病有关，使其成为重要的治疗靶点，但仍未得到充分利用 因为我们对交通运输的机械理解很差。在这个延续中，我们寻求：（一）理解 这种机制的复杂性与各种不同的FG-Nups（具有不同的“风味”的FGs）和不同的 核转运因子（NTR）; ii）建立与潜在的不同 NTR的机制途径，以及如何在NPC中区分NTR和非转运的生物分子; 和iii）开拓如何理解组分的纳米尺度系综结构。我们的综合 使用蛋白质工程，NMR，SANS以及其他生物物理方法和分子动力学进行研究 (MD)模拟提供范例工具验证的IDP系统具有良好的定义（如果不寻常） 功能因此，我们的目标是以原子分辨率剖析FG如何选择性地重复NPC中的区域 限制非特异性大分子的扩散，同时允许NTR的有效交换，使用三个 协同但不重叠的目的：（1）确定不同FG重复序列相互作用的特异性， 风味和NTR类型;（2）确定FG重复序列的整体结构以及它们如何在 与NTR的相互作用;（3）确定不同NTR在不同NTR的集合存在下如何移动。 FG重复口味。 本研究的成果将是第一个FG重复序列功能作用的原子尺度动态图像 区域通过描述两个主要风味的FG重复贡献了NPC中的大部分通量。这是 在三个层次上：i）不同FG重复类型与不同NTR相互作用的主题和变化; ii） 不同FG重复类型的潜在聚合物性质; iii）相对扩散的实际增强如何 与其他类似材料相比，NTR的性能有所提高。这项研究的影响将是巨大的 增加了我们对核运输详细机制的了解。这些数据将使路由器能够 未来的翻译努力，以修改核运输，其中它已受到不利影响的病变细胞。

The sole mediators of nucleocytoplasmic transport of macromolecules are Nuclear Pore Complexes (NPCs), comprised of proteins termed nucleoporins (Nups). The specific mechanism by which these nucleoporins provide selective diffusion of nuclear transport factor (NTR)-linked cargoes is hypothesized to involve relatively weak interactions between transport factors and phenylalanyl glycyl rich (FG) repeat regions found in certain Nups (FG-Nups) to provide selectivity, while the continuous high degree of FG-repeat region dynamic disorder provides sufficient speed to the process, aiding selective diffusion. Defects in nuclear transport and the NPC are associated with numerous diseases, making them an important therapeutic target that is still underused due to our poor mechanistic understanding of transport. In this continuation, we seek to:(i) understand the complexities of this mechanism with various different FG-Nups (with different `flavors' of FGs) and different nuclear transport factors (NTRs); ii) establish the underlying properties associated with potential different mechanistic routes for NTRs, and how NTRs and non-transported biomolecules are discriminated in the NPC; and iii) pioneer how to understand the nanonscale ensemble structures of the components. Our integrative studies using protein engineering, NMR, SANS, as well as other biophysical methods and molecular dynamics (MD) simulation to provide paradigmatic tools validated for an IDP system with a well-defined (if unusual) function. Our goals are therefore to dissect at atomic resolution how FG repeat regions in the NPC selectively restrict diffusion of non-specific macromolecules while permitting the efficient exchange of NTRs, using three synergistic but non-overlapping Aims: (1) determine the specificities of interactions of different FG-repeat flavors and NTR types; (2) determine the ensemble structures of FG repeats and how they are altered on interaction with NTRs; and (3) determine how different NTRs move in the presence of ensembles of different FG repeat flavors. The outcome of this research will be the first atomic scale dynamic pictures of the functional roles of FG repeat regions by describing for the two major flavors of FG repeats contributing to most of the flux in the NPC. This is at three levels: i) themes and variations in the interaction of different FG repeat types with different NTRs; ii) underlying polymer properties of different FG repeat types; iii) how the actual enhancement of relative diffusion of NTRs compared to other similar materials comes about. The impact of this research will be a substantial increase in our knowledge of the detailed mechanisms of nuclear transport. These data will enable a route to future translational efforts to modify nuclear transport where it has been adversely affected in diseased cells.