Abstract:Protein-protein interactions are central to biological processes.
In vitro
methods to examine protein-protein interactions are generally categorized into two classes: in-solution and surface-based methods. Here, using the multivalent interactions between nucleocytoplasmic transport factors and intrinsically disordered FG repeat containing nuclear pore complex proteins as a model system, we examined the utility of three surface-based methods: atomic force microscopy, quartz crystal microb… Show more
“…Gold surfaces coated with Nsp1 or Nsp1-S were characterized with SPR under similar coating conditions as for QCM-D, yielding grafting distances of 4.9 ± 0.1 nm for Nsp1 and 5.8 ± 0.4 nm for Nsp1-S. Upon flushing Kap95, we found, consistent with previous studies 27 , 40 , a concentration-dependent adsorption to Nsp1 brushes (Fig. 2e ), whereas we did not observe any detectable interaction between Kap95 and Nsp1-S (Fig.…”
Section: Resultssupporting
confidence: 89%
“…This minimizes unintentional interactions between Kap95 and gold for subsequent binding experiments (Supplementary Fig. 5 ) 17 , 18 , 40 . Fig.…”
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 selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over 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 selective NPCs.
“…Gold surfaces coated with Nsp1 or Nsp1-S were characterized with SPR under similar coating conditions as for QCM-D, yielding grafting distances of 4.9 ± 0.1 nm for Nsp1 and 5.8 ± 0.4 nm for Nsp1-S. Upon flushing Kap95, we found, consistent with previous studies 27 , 40 , a concentration-dependent adsorption to Nsp1 brushes (Fig. 2e ), whereas we did not observe any detectable interaction between Kap95 and Nsp1-S (Fig.…”
Section: Resultssupporting
confidence: 89%
“…This minimizes unintentional interactions between Kap95 and gold for subsequent binding experiments (Supplementary Fig. 5 ) 17 , 18 , 40 . Fig.…”
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 selective binding of the artificial FG-Nup brushes to the transport receptor Kap95 over 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 selective NPCs.
“…Upon binding of Nsp1 and Nsp1-S to the gold surface under the same conditions as for NupX, we measured a '~5 .8 nm and '~5 .4 nm for Nsp1 and Nsp1-S, respectively, gratifyingly close to the value for NupX. Consistent with previous studies 27, 45 , we found that Kap95 exhibits a concentration-dependent absorption to Nsp1 brushes ( Figure 2e), whereas we did not observe any detectable interaction between Kap95 and Nsp1-S ( Figure 2h).…”
Section: Qcm-d Experiments and MD Simulations Show Selective Bindingsupporting
confidence: 90%
“…Assuming 35% of the sensed film to be constituted by protein mass 43 , with the remaining 65% being constituted by coupled water, we estimated an average grafting distance ' between adjacent NupX proteins of '~5 .6 nm, assuming a triangular lattice (since an equilateral triangulated (hexagonal) lattice is the densest type of packing that can be described by a unique length scale that sets the grafting density). After the Nup-layer was formed, a 1-mercapto-11undecylte-tra(ethyleneglycol) molecule, which is expected to form a ~2 nm thin passivating film 17 , was added to passivate any remaining bare gold that was exposed in between NupX molecules, which minimizes unintentional interactions between Kap95 and gold ( Figure S3) 17,18,45 . After thus setting up a NupX-coated layer, we flushed in Kap95 at stepwise increasing concentrations (~10-1000 nM, Figure 2b) and monitored binding to the NupX-coated surface.…”
Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning
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 ...
“…While important insight was obtained from studies in cellular models [ 11 , 12 , 13 , 14 ], much of our understanding about NPC mediated transport comes from in vitro and in silico (physics-based computational) studies assessing the roles of FG Nup assemblies, NTR binding to FG Nups, effects of NTRs on NPC morphology, and the role of cellular environment in effective transport [ 33 , 34 , 35 , 36 , 37 ]. Additionally, in vitro and in silico NPC (barrier) mimics have been powerful tools for interrogating transport, probing interactions between FG Nups and NTRs, and detailing NPC mediated transport [ 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 ].…”
Specific macromolecules are rapidly transported across the nuclear envelope via the nuclear pore complex (NPC). The selective transport process is facilitated when nuclear transport receptors (NTRs) weakly and transiently bind to intrinsically disordered constituents of the NPC, FG Nups. These two types of proteins help maintain the selective NPC barrier. To interrogate their binding interactions in vitro, we deployed an NPC barrier mimic. We created the stationary phase by covalently attaching fragments of a yeast FG Nup called Nsp1 to glass coverslips. We used a tunable mobile phase containing NTR, nuclear transport factor 2 (NTF2). In the stationary phase, three main factors affected binding: the number of FG repeats, the charge of fragments, and the fragment density. We also identified three main factors affecting binding in the mobile phase: the avidity of the NTF2 variant for Nsp1, the presence of nonspecific proteins, and the presence of additional NTRs. We used both experimentally determined binding parameters and molecular dynamics simulations of Nsp1FG fragments to create an agent-based model. The results suggest that NTF2 binding is negatively cooperative and dependent on the density of Nsp1FG molecules. Our results demonstrate the strengths of combining experimental and physical modeling approaches to study NPC-mediated transport.
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