Spatial structure of disordered proteins dictates conductance and selectivity in nuclear pore complex mimics

Ananth, Adithya N.; Mishra, Ankur; Frey, Steffen; Dwarkasing, Arvind; Versloot, Roderick Corstiaan Abraham; Giessen, E. van der; Görlich, Dirk; Onck, Patrick; Dekker, Cees

doi:10.7554/elife.31510

Cited by 45 publications

(146 citation statements)

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“…To assess the size-selective properties of the NupX brushes, we performed umbrella sampling simulations of the absorption of Kap95 and tCherry to NupX brushes (Materials and Methods). We modelled Kap95 ( Figure S14) as an 8.5 nm sized sterically repulsive (i.e., modelling only repulsive, excluded volume interactions) particle with 10 hydrophobic binding sites 8,31,48,49 and a total charge density similar to that of Kap95 (-43e). tCherry was modelled as a sterically inert spherical particle of 7.5 nm diameter 10 .…”

Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 99%

“…These NPC properties complicate in-vivo studies 3,21-23 , for which it is very challenging to identify contributions coming from individual FG-Nups 24,25 . On the other hand, in-vitro approaches to study nucleocytoplasmic transport using biomimetic NPC systems [26][27][28][29][30][31][32][33] have thus far been limited to single native FG-Nups and mutations thereof, attempting to understand the physical behaviour of FG-Nups and their interactions with NTRs. The reliance on a few selected Nups from yeast or humans in these studies with sequences that evolved over time in different ways for each of these specific organisms makes it difficult to pinpoint the essential and minimal properties that provide FG-Nups with their specific selective functionality.Here, we describe a bottom-up approach to studying nuclear transport selectivity, where we rationally design, synthesize, and assess artificial FG-Nups with user-defined properties that are set by an amino acid sequence that is chosen by the user.…”

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confidence: 99%

“…Secondly, we explore the transport properties of NupX-functionalized solid-state nanopores and show that NupXlined pores constitute a selective barrier. Similar to FG-Nups previously studied with the same technique 28,31 , the NPC-mimicking nanopores allow fast and efficient passage of Kap95 molecules, while blocking transport of BSA. Coarse-grained MD simulations of NupX-functionalized nanopores highlight the formation of a dense FG-rich meshwork with similar protein densities as in native NPCs, which excludes tCherry but allows entry and passage of Kap95.…”

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confidence: 99%

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

Fragasso

Vries

Sluis

et al. 2020

Preprint

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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 ...

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Section: Qcm-d Experiments and MD Simulations Show Selective Bindingmentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

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

Fragasso

Vries

Sluis

et al. 2020

Preprint

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“…Thus, the macroscopic dimensions of the instruments could well overshadow the molecular interactions and diffusion effects that are biologically relevant at nanoscopic scales. Solution studies in vitro, such as ITC, stopped-flow kinetics, NMR, and appropriately-scaled NPC mimics such as nanoscopic pores [48,[80][81][82] and DNA origami pores [83,84] offer the possibility of studying FG-TF interactions at a scale more commensurate with that of nuclear transport in vivo.…”

Section: Macroscopic Instruments Versus Nanoscopic Biological Systemsmentioning

confidence: 99%

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

Hayama

Sorci

Keating

et al. 2018

Preprint

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BTC), and rout@mail.rockefeller.edu (MPR) * These authors contributed equally to this work.Keywords: Quartz crystal microbalance with dissipation (QCM-D), surface plasmon resonance (SPR), atomic force microscopy (AFM), mass transport limitation, protein-protein interaction, multivalency, intrinsically disordered protein (IDP), nuclear pore complex (NPC), FG nucleoporin (FG Nup), nucleocytoplasmic transport. 2 AbstractProtein-protein interactions are central to biological processes and the methods to thoroughly characterize them are of great interest. 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 involved in nucleocytoplasmic transport as a model system, we examined the utility of three surface-based methods in characterizing rapid interactions involving intrinsically disordered proteins: atomic force microscopy, quartz crystal microbalance with dissipation, and surface plasmon resonance. Although results were comparable to those of previous reports, the apparent effect of mass transport limitations was demonstrated. Additional experiments with a loss-of-interaction mutant variant demonstrated the existence of additional physical phenomena and an uncharacterized binding mode. These results indicate the binding events that take place on the surface can be quite complex, suggesting particular care must be exercised in interpretation of such data.Recently, we and others have reported in-solution affinities between TFs and individual FG motifs, whose per-FG-motif KDs were in the millimolar range, compatible with the rapid kinetics of TF translocation through the NPC [17][18][19]. We also found that multiple low-affinity interactions can yield a higher overall interaction specificity than monovalent ones without compromising a high on-off rate of individual FG motifs [19]. Thus, one motivation of this work was to investigate the cause of these discrepancies in affinity measurements conducted by various methods.As outlined by Schuck and Zhao, analysis of multivalent interactions by surface-based methods require extra care because of potential complexities of the binding mechanism on the surface; in some cases rendering the results "impossible to realistically interpret" [35]. In addition, (i) it is often difficult to quantify the amount or the density of protein conjugated to the surface; (ii) conjugated surfaces usually have an inhomogeneous distributions of ligands [35]; (iii) the degree and the effect of analyte retention on the surface after a binding experiment is often not assessed; (iv) mass transport limitations can significantly affect measurements of binding kinetics when using SPR and QCM-D, and are often overlooked [35][36][37]; (v) change in protein conformation or denaturation upon binding to surfaces can occur [38,39], and (vi) macroscopic effects of surface crowding, especially in the context of multivalency, are not trivial to adequately address and quantify for surface-...

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“…These problems can be overcome by using experimentally-calibrated coarse-grained molecular dynamics (MD) models that can capture the sequence specificity and are suitable for simulating high-density phases of proteins (37)(38)(39). In this study we use our one-bead-peramino-acid (1BPA) coarse-grained MD model (37,40,41)…”

Section: Introductionmentioning

confidence: 99%

Phase separation of toxic dipeptide repeat proteins related to C9orf72 ALS/FTD

Jafarinia

Giessen

Onck

2020

Preprint

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The expansion mutation in the C9orf72 gene is the most common known genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation can produce five dipeptide repeat proteins (DPRs), of which three are known to be toxic: poly-PR, poly-GR, and poly-GA. The toxicity of poly-GA is attributed to its aggregation in the cytoplasm, while for poly-PR and poly-GR several toxicity pathways have been proposed. The toxicity of the DPRs has been shown to depend on their length, but the underlying molecular mechanism of this length dependence is not well understood. To address this, a one-bead-per-amino-acid (1BPA) coarse-grained molecular dynamics model is used to study the single-molecule and phase separation properties of the DPRs. We find a strong dependence of the phase separation behavior on both DPR length and concentration, with longer DPRs having a higher propensity to phase separate and form condensed phases with higher concentrations. The critical lengths required for phase separation (25 for poly-PR and 50 for poly-GA) are comparable to the toxicity threshold limit of 30 repeats found for the expansion mutation in patient cells, suggesting that phase separation could play an important role in DPR toxicity. ALS/FTD | DPRs | single-molecule properties | phase separationCorrespondence: p.r.onck@rug.nl

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Spatial structure of disordered proteins dictates conductance and selectivity in nuclear pore complex mimics

Cited by 45 publications

References 56 publications

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

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

Interactions of nuclear transport factors and surface-conjugated FG nucleoporins: Insights and limitations

Phase separation of toxic dipeptide repeat proteins related to C9orf72 ALS/FTD

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