The dynamic mechanism of nuclear transport visualized at the atomic scale

大分子核细胞质运输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。这些核孔蛋白的具体机制 假设核转运因子（NTR）相关货物的选择性扩散涉及相对 某些转运因子与富含苯丙氨酰甘氨酰 (FG) 的重复区域之间的相互作用较弱 Nups（FG-Nups）提供选择性，同时连续高度的FG-repeat区域动态无序 为该过程提供足够的速度，有助于选择性扩散。核运输和 NPC 的缺陷 与许多疾病相关，使其成为尚未得到充分利用的重要治疗靶点 由于我们对运输的机械理解很差。在此续篇中，我们力求：(i) 了解 该机制与各种不同的 FG-Nups（具有不同“风味”的 FG）和不同的 FG-Nups 的复杂性 核转运因子（NTR）； ii) 建立与潜在差异相关的基本属性 NTR 的机制途径，以及 NPC 中如何区分 NTR 和非转运生物分子； iii) 开创了如何理解组件的纳米级系综结构的先河。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，为具有明确定义（如果不常见）的 IDP 系统提供经过验证的范例工具 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，使用三种 协同但不重叠的目标：（1）确定不同FG-重复序列相互作用的特异性 口味和 NTR 类型； (2) 确定 FG 重复的整体结构以及它们是如何改变的 与 NTR 的互动； (3) 确定不同 NTR 在存在不同类型的集合的情况下如何移动 FG重复口味。 这项研究的成果将是首张原子尺度动态图片，展示 FG 重复序列的功能作用 通过描述对 NPC 中大部分通量有贡献的 FG 重复的两种主要类型来描述区域。这是 三个层面：i) 不同 FG 重复类型与不同 NTR 相互作用的主题和变化；二） 不同 FG 重复类型的基本聚合物特性； iii) 相对扩散的实际增强如何 NTR 与其他类似材料相比的结果。这项研究的影响将是巨大的 增加我们对核运输详细机制的了解。这些数据将启用一条路线 未来的转化努力是为了改变在患病细胞中受到不利影响的核运输。

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.