The dynamic mechanism of nuclear transport visualized at the atomic scale

大分子核质运输的唯一媒介是核孔复合体(NPC)， 由名为核孔素(NUPS)的蛋白质组成。这些核孔素的具体机制 提供与核运输因子(NTR)相关的货物的选择性扩散假设涉及相对 转运因子与某些富含苯丙氨基甘氨酰(FG)重复序列的弱相互作用 NUPS(FG-NUPS)提供选择性，同时持续高度的FG-重复区域动态无序 为这一过程提供了足够的速度，有助于选择性扩散。核运输中的缺陷与全国人大 与许多疾病有关，使其成为一个重要的治疗靶点，但仍未得到充分利用 这是因为我们对运输的机械理解很差。在本文中，我们力求：(I)了解 对于不同的FG-nup(具有不同的FG‘味道’)和不同的FG-nup，这种机制的复杂性 核运输因素(NTR)；二)建立与潜在不同 非关税壁垒的机械路线，以及在全国人大中如何区分非关税壁垒和未运输的生物分子； 以及iii)开拓性地了解组件的纳米级系综结构。我们的一体化 利用蛋白质工程、核磁共振、三聚氰胺以及其他生物物理方法和分子动力学进行研究 (MD)模拟，以提供针对IDP系统进行验证的范例工具，且定义明确(如果不常见) 功能。因此，我们的目标是在原子分辨率下剖析FG如何选择性地重复NPC中的区域 限制非特定大分子的扩散，同时允许有效交换非特定受体，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 风味和NTR类型；(2)确定FG重复序列的集合结构及其如何改变 与非关税壁垒的互动；以及(3)确定不同的非关税壁垒如何在存在不同的非关税壁垒的情况下移动 FG重复口味。 这项研究的结果将是FG重复功能的第一张原子尺度动态图像 通过描述FG重复的两种主要口味来描述区域，这些重复对NPC中的大部分通量做出了贡献。这是 在三个层面上：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)实际如何增强相对扩散 与其他类似材料相比，非关税壁垒产生了更大的影响。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。这些数据将使一条通往 在疾病细胞中受到不利影响的情况下，未来修改核运输的翻译努力。

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.