Converging on the function of intrinsically disordered nucleoporins in the nuclear pore complex

Several biological mechanisms involve proteins or proteinaceous components that are intrinsically disordered. A case in point pertains to the nuclear pore complex (NPC), which regulates molecular transport between the nucleus and the cytoplasm. NPC functionality is dependent on unfolded domains rich in Phe-Gly (FG) repeats (i.e., FG-domains) that collectively act to promote or hinder cargo translocation. To a large extent, our understanding of FG-domain behavior is limited to in vitro investigations given the difficulty to resolve them directly in the NPC. Nevertheless, recent findings indicate a collective convergence towards rationalizing FG-domain function. This review aims to glean further insight into this fascinating problem by taking an objective look at the boundary conditions and contextual details underpinning FG-domain behavior in the NPC. Here, we treat the FG-domains as being commensurate with polymeric chains to address ambiguities such as for instance, how FG-domains tethered to the central channel of the NPC would behave differently as compared with their free-floating counterparts in solution. By bringing such fundamental questions to the fore, this review seeks to illuminate the importance of how such parameters can hold influence over the structure-function relation of intrinsically disordered proteins in the NPC and beyon

Karyopherins regulate nuclear pore complex barrier and transport function

Nucleocytoplasmic transport is sustained by karyopherins (Kaps) and a Ran guanosine triphosphate (RanGTP) gradient that imports nuclear localization signal (NLS)–specific cargoes (NLS-cargoes) into the nucleus. However, how nuclear pore complex (NPC) barrier selectivity, Kap traffic, and NLS-cargo release are systematically linked and simultaneously regulated remains incoherent. In this study, we show that Kap α facilitates Kap β 1 turnover and occupancy at the NPC in a RanGTP-dependent manner that is directly coupled to NLS-cargo release and NPC barrier function. This is underpinned by the binding affinity of Kap β 1 to phenylalanine–glycine nucleoporins (FG Nups), which is comparable with RanGTP·Kap β 1, but stronger for Kap α ·Kap β 1. On this basis, RanGTP is ineffective at releasing standalone Kap β 1 from NPCs. Depleting Kap α ·Kap β 1 by RanGTP further abrogates NPC barrier function, whereas adding back Kap β 1 rescues it while Kap β 1 turnover softens it. Therefore, the FG Nups are necessary but insufficient for NPC barrier function. We conclude that Kaps constitute integral constituents of the NPC whose barrier, transport, and cargo release functionalities establish a continuum under a mechanism of Kap-centric control

Gate-Crashing the Nuclear Pore Complex

As a third in a series of MD simulations investigating the binding dynamics between nuclear transport receptors and FG-repeats, Isgro and Schulten (2007b) unveil that close, physical intimacy between partners is likely to ensure a hassle-free passage through the nuclear pore complex

In Silico Access to the Nuclear Pore Complex

Translocation of biomolecules through the nuclear pore complex is governed by interactions that occur between phenylalanine-glycine-rich nucleoporins and transport receptors. Using molecular dynamics simulations, Isgro and Schulten (2005) replicate and predict these interactions with startling spatial clarity and temporal detail

Karyopherin enrichment at the nuclear pore complex attenuates Ran permeability

Ran is a small GTPase whose nucleotide-bound forms cycle through nuclear pore complexes (NPCs) to direct nucleocytoplasmic transport (NCT). Generally, Ran guanosine triphosphate (GTP) binds cargo-carrying karyopherin receptors (Kaps) in the nucleus and liberates them in the cytoplasm following hydrolysis to Ran guanosine diphosphate (GDP). This generates a remarkably steep Ran gradient across the nuclear envelope that sustains compartment-specific cargo delivery and accumulation. However, because NPCs are permeable to small molecules of comparable size, it is unclear how an uncontrolled mixing of RanGTP and RanGDP is prevented. Here, we find that an NPC-enriched pool of Kapβ1 selectively mediates Ran diffusion across the pore, but not passive molecules of similar size (e.g. GFP). This is due to binding interactions with Kapβ1, which is stronger for RanGTP, but weaker for RanGDP. On this basis, the RanGDP importer, nuclear transport factor 2 (NTF2), facilitates the release of RanGDP from Kapβ1 following GTP hydrolysis. Accordingly, the enrichment of Kapβ1 at NPCs may function as a retention mechanism that preserves the sharp transition of RanGTP and RanGDP in the nucleus and cytoplasm, respectively.</jats:p

Helium Scanning Transmission Ion Microscopy and Electrical Characterization of Glass Nanocapillaries with Reproducible Tip Geometries

Nanopores fabricated from glass microcapillaries are used in applications ranging from scanning ion conductance microscopy to single-molecule detection. Still, evaluating the nanocapillary tip by a noninvasive means remains challenging. For instance, electron microscopy characterization techniques can charge, heat, and contaminate the glass surface and typically require conductive coatings that influence the final tip geometry. <i>Per contra</i>, electrical characterization by the means of ion current through the capillary lumen provides only indirect geometrical details of the tips. Here, we show that helium scanning transmission ion microscopy provides a nondestructive and precise determination of glass nanocapillary tip geometries. This enables the reproducible fabrication of axially asymmetric blunt, bullet, and hourglass-shaped tips with opening diameters from 20 to 400 nm by laser-assisted pulling. Accordingly, this allows for an evaluation of how tip shape, pore diameter, and opening angle impact ionic current rectification behavior and the translocation of single molecules. Our analysis shows that current drops and translocation dwell times are dominated by the pore diameter and opening angles regardless of nanocapillary tip shape

Promiscuous Binding of Karyopherinβ1 Modulates FG Nucleoporin Barrier Function and Expedites NTF2 Transport Kinetics

AbstractThe transport channel of nuclear pore complexes (NPCs) contains a high density of intrinsically disordered proteins that are rich in phenylalanine-glycine (FG)-repeat motifs (FG Nups). The FG Nups interact promiscuously with various nuclear transport receptors (NTRs), such as karyopherins (Kaps), that mediate the trafficking of nucleocytoplasmic cargoes while also generating a selectively permeable barrier against other macromolecules. Although the binding of NTRs to FG Nups increases molecular crowding in the NPC transport channel, it is unclear how this impacts FG Nup barrier function or the movement of other molecules, such as the Ran importer NTF2. Here, we use surface plasmon resonance to evaluate FG Nup conformation, binding equilibria, and interaction kinetics associated with the multivalent binding of NTF2 and karyopherinβ1 (Kapβ1) to Nsp1p molecular brushes. NTF2 and Kapβ1 show different long- and short-lived binding characteristics that emerge from varying degrees of molecular retention and FG repeat binding avidity within the Nsp1p brush. Physiological concentrations of NTF2 produce a collapse of Nsp1p brushes, whereas Kapβ1 binding generates brush extension. However, the presence of prebound Kapβ1 inhibits Nsp1p brush collapse during NTF2 binding, which is dominated by weak, short-lived interactions that derive from steric hindrance and diminished avidity with Nsp1p. This suggests that binding promiscuity confers kinetic advantages to NTF2 by expediting its facilitated diffusion and reinforces the proposal that Kapβ1 contributes to the integral barrier function of the NPC

Karyopherin-Centric Control of Nuclear Pores Based on Molecular Occupancy and Kinetic Analysis of Multivalent Binding with FG Nucleoporins

AbstractIntrinsically disordered Phe-Gly nucleoporins (FG Nups) within nuclear pore complexes exert multivalent interactions with transport receptors (Karyopherins (Kaps)) that orchestrate nucleocytoplasmic transport. Current FG-centric views reason that selective Kap translocation is promoted by alterations in the barrier-like FG Nup conformations. However, the strong binding of Kaps with the FG Nups due to avidity contradicts rapid Kap translocation in vivo. Here, using surface plasmon resonance, we innovate a means to correlate in situ mechanistic (molecular occupancy and conformational changes) with equilibrium (binding affinity) and kinetic (multivalent binding kinetics) aspects of Karyopherinβ1 (Kapβ1) binding to four different FG Nups. A general feature of the FxFG domains of Nup214, Nup62, and Nup153 is their capacity to extend and accommodate large numbers of Kapβ1 molecules at physiological Kapβ1 concentrations. A notable exception is the GLFG domain of Nup98, which forms a partially penetrable cohesive layer. Interestingly, we find that a slowly exchanging Kapβ1 phase forms an integral constituent within the FG Nups that coexists with a fast phase, which dominates transport kinetics due to limited binding with the pre-occupied FG Nups at physiological Kapβ1 concentrations. Altogether, our data reveal an emergent Kap-centric barrier mechanism that may underlie mechanistic and kinetic control in the nuclear pore complex