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April 24th, 2025
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Zernike Institute for Advanced Materials, University of Groningen
biophysics
biorxiv

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

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

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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