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

This is the authors' abstract. We don't add key points for this paper.

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 such as the karyopherin (Kap) family of proteins. How the amino acid sequence of FG-Nups contributes to this selectivity 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 yeast GLFG-Nups), we design, synthesize, and experimentally test a library of NupY variants using quartz crystal microbalance with dissipation monitoring 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 variants and the highest transport selectivity for designs close to native GLFG-Nups. We postulate that a balance between steric hindrance and enthalpic gain from multivalent Kap-FG-Nup interactions drives the spatial and temporal partitioning of Kaps in the NPC.