Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

de Vries, H. W.; Barth, A.; Fragasso, A.; Otto, T. A.; van der Graaf, A.; van der Sluis, E. O.; van der Giessen, E.; Veenhoff, L. M.; Dekker, C.; Onck, P. R.

doi:10.1101/2025.04.21.649908

bioRxiv · 10.1101/2025.04.21.649908

Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins

de Vries, H. W.·Barth, A.·Fragasso, A.·Otto, T. A.·van der Graaf, A.·van der Sluis, E. O.·van der Giessen, E.·Veenhoff, L. M.·Dekker, C.·Onck, P. R.

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Abstract

Nucleocytoplasmic transport occurs via nuclear pore complexes (NPCs), [~]40-60 nm wide pores lined with intrinsically disordered proteins that are rich in Phe-Gly motifs (FG-Nups) that form a selective barrier. Molecules larger than [~]50 kDa are increasingly blocked for transport unless they are bound to a nuclear transport receptor (NTR). How the amino acid sequence of FG-Nups contribute to this is not fully understood. Here, we present de novo designed artificial FG-Nups with a systematically varied FG-repeat spacing and charge-to-hydrophobicity ratio (C/H). Starting from a reference sequence termed NupY (with the average properties of natural yeast GLFG- Nups), we designed, synthesized, and experimentally tested a library of NupY variants using QCM-D experiments and phase separation assays. We find that the spacing between FG-motifs governs Kap95 absorption into the FG- Nup phase, while increasing C/H results in higher avidity for Kap95 due to an increased accessibility of FG-motifs. Molecular dynamics simulations of transport through NupY-coated pores show a reduced barrier function for noncohesive high-C/H-ratio variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between entropic repulsion and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.

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de Vries, H. W., Barth, A., Fragasso, A., Otto, T. A., van der Graaf, A., van der Sluis, E. O., van der Giessen, E., Veenhoff, L. M., Dekker, C., Onck, P. R.. 2025-04-24. Spatial control of karyopherin binding avidity within NPC mimics revealed by designer FG-Nucleoporins. https://doi.org/10.1101/2025.04.21.649908

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