A designer FG-Nup that reconstitutes the selective transport barrier of the Nuclear Pore Complex

Abstract

Nuclear Pore Complexes (NPCs) regulate bidirectional transport between the nucleus and the cytoplasm. Intrinsically disordered FG-Nups line the NPC lumen and form a selective barrier, where transport of most proteins is inhibited whereas specific transporter proteins freely pass. The mechanism underlying selective transport through the NPC is still debated. Here, we reconstitute the selective behaviour of the NPC bottom-up by introducing a rationally designed artificial FG-Nup that mimics natural Nups. Using QCM-D, we measure a strong affinity of the artificial FG-Nup brushes to the transport receptor Kap95, whereas no binding occurs to cytosolic proteins such as BSA. Solid-state nanopores with the artificial FG-Nups lining their inner walls support fast translocation of Kap95 while blocking BSA, thus demonstrating selectivity. Coarse-grained molecular dynamics simulations highlight the formation of a selective meshwork with densities comparable to native NPCs. Our findings show that simple design rules can recapitulate the selective behaviour of native FG-Nups and demonstrate that no specific spacer sequence nor a spatial segregation of different FG-motif types are needed to create functional NPCs. IntroductionNucleocytoplasmic transport is orchestrated by the Nuclear Pore Complex (NPC), which imparts a selective barrier to biomolecules 1,2 . The NPC is a large eightfold-symmetric protein complex (with a size of ~52 MDa in yeast and ~112 MDa in vertebrates) that is embedded within the nuclear envelope and comprises ~30 different types of Nucleoporins ('Nups') 3,4 . Intrinsically disordered proteins, termed FG-Nups, line the central channel of the NPC. FG-Nups are characterized by the presence of phenylalanine-glycine (FG) repeats separated by spacer sequences 5 and they are highly conserved throughout species 6 . FG-Nups carry out a dual function: By forming a dense barrier (100-200 mg/mL) within the NPC lumen, they allow passage of molecules in a size-selective manner 7-10 . Small molecules can freely diffuse through, whereas larger particles are generally excluded 11 . At the same time, FG-Nups mediate the transport of large NTR-bound (Nuclear Transport Receptor) cargoes across the NPC through transient hydrophobic interactions between FG repeats and hydrophobic pockets on the convex side of NTRs 12 . Various models have been developed in order to connect the physical properties of FG-Nups to the size-selective properties of the NPC central channel, e.g. the 'virtual-gate' 13 , 'selective phase' 14,15 , 'reduction of dimensionality' 16 , 'kap-centric' 17-19 , 'polymer brush' 20 , and 'forest' 5 models.As is evident from the multitude of transport models, no consensus on the NPC transport mechanisms has yet been reached.The NPC is highly complex in its architecture and dynamics, being constituted by many different Nups that simultaneously interact with multiple transiting cargoes and NTRs. In fact, translocating cargoes may amount to almost half of the mass of the central channel, so they may be considered an ...

Free energy calculations shed light on the nuclear pore complex’s selective barrier nature

“…From the dynamical point of view, atomic force microscopy measurements (29) showed that FG-Nups fluctuate rapidly inside the nuclear pore. Molecular dynamics (30,31), Langevin dynamics (32)(33)(34)(35)(36), and Brownian dynamics simulations (37)(38)(39)(40)(41) demonstrated the highly dynamic nature of FG-Nups. However, some experimental studies argued that the entropic effect by itself is too weak to account for the mechanical stiffness in the NPC central channel (42,43).…”

“…To investigate the relation between the entropic barrier effect and the NPC's selective nature, we modeled the free energy by specifically considering the conformational entropy of FG-Nups and the binding interaction between FG-Nups and NTRs. Other groups have previously proposed the models for the NPC's free energy using the molecular theory (51)(52)(53)(54)(55), the density functional theory (56,57), and umbrella sampling of the molecular dynamics simulation trajectories (33,36). Our model is different from prior art in two ways: firstly, we assumed that FG-Nups are homogeneous ideal chains instead of considering their amino acid sequence heterogeneity (33,36,52,54) or intermolecular cohesiveness (56,57).…”

“…Other groups have previously proposed the models for the NPC's free energy using the molecular theory (51)(52)(53)(54)(55), the density functional theory (56,57), and umbrella sampling of the molecular dynamics simulation trajectories (33,36). Our model is different from prior art in two ways: firstly, we assumed that FG-Nups are homogeneous ideal chains instead of considering their amino acid sequence heterogeneity (33,36,52,54) or intermolecular cohesiveness (56,57). By this reductionist approach, we focused on studying the effect of FG-Nups' conformational entropy in absence of other physical factors.…”

Molecular basis of C9orf72 poly-PR interference with the β-karyopherin family of nuclear transport receptors

“…Although these studies provide interesting insights into potential pathways for R-DPRs-mediated NCT defects, the molecular basis for the interaction of longer R-DPRs with different Kapβs and the resulting NCT defects has not yet been identified. In this study, we use coarse-grained (CG) molecular dynamics models [33][34][35][36][37] to investigate the length-dependent interaction of poly-PR (known to be the most toxic DPR [11][12][13]) with different members of the Kapβ family of NTRs with the aim to provide mechanistic insight into the way poly-PR interferes with the transport functionality of Kapβs.…”

In and through the membrane by solid-state NMR: investigations of the human voltage-dependent anion channel 1 and Nup98-derived FG-domains

“…Designer FG-domains, where the composition of the sequence mirrors that of the wild-type proteins (Fragasso et al, 2020) provide an excellent opportunity for constructing sequences amenable to atomic resolution characterization by NMR. Through simplification of the sequence of Tetrahymena thermophila MacNup98A (TtMacNup98A), Ng and Görlich obtained a perfectly repetitive sequence that could entirely recapitulate the permselectivity properties of the full-length protein and of nuclear pores (Ng and Görlich, 2021) (unpublished).…”