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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.


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Transport receptor occupancy in nuclear pore complex mimics

Show Author's information Alessio Fragasso1,§Hendrik W. de Vries2,§John Andersson3Eli O. van der Sluis1Erik van der Giessen2Patrick R. Onck2( )Cees Dekker1( )
Department of Bionanoscience, Kavli Institute of Nanoscience, Delft University of Technology, Van der Maasweg 9, Delft 2629 HZ, The Netherlands
Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, Groningen 9747AG, The Netherlands
Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Kemivägen 10, Gothenburg SE-412 96, Sweden

§ Alessio Fragasso and Hendrik W. de Vries contributed equally to this work.

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.

Keywords:

nuclear pore complex, intrinsically disordered proteins, nuclear transport receptors, karyopherins, nanopores, biomimetics, molecular dynamics, coarse-grained modeling
Received: 21 December 2021 Revised: 03 June 2022 Accepted: 06 June 2022 Published: 01 July 2022 Issue date: November 2022
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Received: 21 December 2021
Revised: 03 June 2022
Accepted: 06 June 2022
Published: 01 July 2022
Issue date: November 2022

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© The Author(s) 2022

Acknowledgements

Acknowledgements

We would like to thank the Görlich Lab for sharing purified Nsp1, Meng-yue Wu for technical assistance on the TEM, and Marc op den Kamp, Sjoerd Meesters, and Koen Wortelboer for their assistance in developing precursors to the Kap95 model. This research was funded by NWO-I programme “Projectruimte”, Grant No. 16PR3242-1. We acknowledge discussions with Nils Klughammer, Paola de Magistris, Anders Barth, Adithya Ananth, Sonja Schmid, Hamid Jafarinia, and Mark Driver. H. W. d. V. acknowledges support from the CIT of the University of Groningen and the Berendsen Centre for Multiscale Modeling for providing access to the Peregrine and Nieuwpoort high performance computing clusters. C. D. acknowledges support from the ERC Advanced Grant No. 883684 and the NanoFront and BaSyC programmes.

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