The dynamic mechanism of nuclear transport visualized at the atomic scale

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PARENT AWARD ABSTRACT 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 underuseddue to our poor mechanistic understanding of transport. In this continuation, we seek to:(i) understand the complexities of this mechanism with various 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 nanoscale 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 repeatregions by describing for the two major flavors of FG repeats contributing to most of the flux in the NPC. This isat 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 diffusionof 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.