@article{Fragasso2022, 
author = {Alessio Fragasso and Hendrik W. de Vries and John Andersson and Eli O. van der Sluis and Erik van der Giessen and Patrick R. Onck and Cees Dekker},
title = {Transport receptor occupancy in nuclear pore complex mimics},
year = {2022},
journal = {Nano Research},
volume = {15},
number = {11},
pages = {9689-9703},
keywords = {molecular dynamics, nanopores, biomimetics, nuclear pore complex, intrinsically disordered proteins, nuclear transport receptors, karyopherins, coarse-grained modeling},
url = {https://www.sciopen.com/article/10.1007/s12274-022-4647-1},
doi = {10.1007/s12274-022-4647-1},
abstract = {Nuclear pore complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG-Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess “Kap-centric” NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.}
}