scholarly journals Transport receptor occupancy in Nuclear Pore Complex mimics

2021 ◽  
Author(s):  
Alessio Fragasso ◽  
Hendrik W. de Vries ◽  
John Andersson ◽  
Eli O. van der Sluis ◽  
Erik van der Giessen ◽  
...  
Keyword(s):  
Ionic Current ◽  
Molecular Transport ◽  
Pore Wall ◽  
Receptor Occupancy ◽  
Coarse Grained ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Intrinsically Disordered ◽  
Noise Data ◽  
Transport Receptor

Nuclear Pore Complexes (NPCs) regulate all molecular transport between the nucleus and the cytoplasm in eukaryotic cells. Intrinsically disordered Phe-Gly nucleoporins (FG Nups) line the central conduit of NPCs to impart a selective barrier where large proteins are excluded unless bound to a transport receptor (karyopherin; Kap). Here, we assess 'Kap-centric' NPC models, which postulate that Kaps participate in establishing the selective barrier. We combine biomimetic nanopores, formed by tethering Nsp1 to the inner wall of a solid-state nanopore, with coarse-grained modeling to show that yeast Kap95 exhibits two populations in Nsp1-coated pores: one population that is transported across the pore in milliseconds, and a second population that is stably assembled within the FG mesh of the pore. Ionic current measurements show a conductance decrease for increasing Kap concentrations and noise data indicate an increase in rigidity of the FG-mesh. Modeling reveals an accumulation of Kap95 near the pore wall, yielding a conductance decrease. We find that Kaps only mildly affect the conformation of the Nsp1 mesh and that, even at high concentrations, Kaps only bind at most 8% of the FG-motifs in the nanopore, indicating that Kap95 occupancy is limited by steric constraints rather than by depletion of available FG-motifs. Our data provide an alternative explanation of the origin of bimodal NPC binding of Kaps, where a stable population of Kaps binds avidly to the NPC periphery, while fast transport proceeds via a central FG-rich channel through lower affinity interactions between Kaps and the cohesive domains of Nsp1.

scholarly journals A designer FG-Nup that reconstitutes the selective transport barrier of the nuclear pore complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Alessio Fragasso ◽  
Hendrik W. de Vries ◽  
John Andersson ◽  
Eli O. van der Sluis ◽  
Erik van der Giessen ◽  
...  
Keyword(s):  
Coarse Grained ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transport Barrier ◽  
Selective Transport ◽  
Intrinsically Disordered ◽  
Bidirectional Transport ◽  
Dynamics Simulations ◽  
Pore Complexes ◽  
Transport Receptor

AbstractNuclear 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.

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

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Adithya N Ananth ◽  
Ankur Mishra ◽  
Steffen Frey ◽  
Arvind Dwarkasing ◽  
Roderick Versloot ◽  
...  
Keyword(s):  
Molecular Transport ◽  
Coarse Grained ◽  
Disordered Proteins ◽  
Striking Difference ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Distribution ◽  
Intrinsically Disordered ◽  
Transport Receptors ◽  
Dynamics Simulations

Nuclear pore complexes (NPCs) lined with intrinsically disordered FG-domains act as selective gatekeepers for molecular transport between the nucleus and the cytoplasm in eukaryotic cells. The underlying physical mechanism of the intriguing selectivity is still under debate. Here, we probe the transport of ions and transport receptors through biomimetic NPCs consisting of Nsp1 domains attached to the inner surface of solid-state nanopores. We examine both wildtype FG-domains and hydrophilic SG-mutants. FG-nanopores showed a clear selectivity as transport receptors can translocate across the pore whereas other proteins cannot. SG mutant pores lack such selectivity. To unravel this striking difference, we present coarse-grained molecular dynamics simulations that reveal that FG-pores exhibit a high-density, nonuniform protein distribution, in contrast to a uniform and significantly less-dense protein distribution in the SG-mutant. We conclude that the sequence-dependent density distribution of disordered proteins inside the NPC plays a key role for its conductivity and selective permeability.

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

2020 ◽  
Author(s):  
Alessio Fragasso ◽  
Hendrik W. de Vries ◽  
Eli O. van der Sluis ◽  
Erik van der Giessen ◽  
Patrick R. Onck ◽  
...  
Keyword(s):  
Coarse Grained ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transport Barrier ◽  
Selective Transport ◽  
Intrinsically Disordered ◽  
Bidirectional Transport ◽  
Dynamics Simulations ◽  
Pore Complexes ◽  
Transport Receptor

AbstractNuclear 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.

Modeling the nucleoporins that form the hairy pores

2020 ◽  
Vol 48 (4) ◽  
pp. 1447-1461 ◽  
Author(s):  
Kai Huang ◽  
Igal Szleifer
Keyword(s):  
State Of The Art ◽  
Mean Field ◽  
Molecular Transport ◽  
Field Theories ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Intrinsically Disordered ◽  
Selective Permeability ◽  
Pore Complexes

Sitting on the nuclear envelope, nuclear pore complexes (NPCs) control the molecular transport between the nucleus and the cytoplasm. Without definite open or close states, the NPC uses a family of intrinsically disordered nucleoporins called FG-Nups to construct a selective permeability barrier whose functional structure is unclear. Experimental advances have offered high-resolution molecular knowledge of the NPC scaffold and docking of the unfolded FG-Nups, however, the ‘hairy’ barrier structure still appears as blurred lobes even under the state-of-the-art microscopy. Without accurate experimental visualization, the molecular mechanism for the NPC-mediated transport remains a matter of debate. Modeling provides an alternative way to resolve this long-standing mystery. Here, we briefly review different methods employed in modeling the FG-Nups, arranging from all-atom molecular dynamics to mean-field theories. We discuss the advantage and limit of each modeling technique, and summarize the theoretical insights that, despite certain controversy, deepened our understanding of the hairy pore.

scholarly journals The molecular mechanism of nuclear transport revealed by atomic-scale measurements

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Loren E Hough ◽  
Kaushik Dutta ◽  
Samuel Sparks ◽  
Deniz B Temel ◽  
Alia Kamal ◽  
...  
Keyword(s):  
Intrinsically Disordered Proteins ◽  
Atomic Scale ◽  
Disordered Proteins ◽  
Nuclear Pore ◽  
Resonance Spectroscopy ◽  
Nuclear Pore Complexes ◽  
Selective Filter ◽  
Intrinsically Disordered ◽  
Cellular Environment ◽  
Pore Complexes

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC.

scholarly journals Size-dependent leak of soluble and membrane proteins through the yeast nuclear pore complex

2015 ◽  
Vol 26 (7) ◽  
pp. 1386-1394 ◽  
Author(s):  
Petra Popken ◽  
Ali Ghavami ◽  
Patrick R. Onck ◽  
Bert Poolman ◽  
Liesbeth M. Veenhoff
Keyword(s):  
Molecular Dynamics ◽  
Membrane Proteins ◽  
Coarse Grained ◽  
Nuclear Pore ◽  
Permeability Barrier ◽  
Nuclear Pore Complexes ◽  
Structural And Functional Properties ◽  
Dynamics Simulations ◽  
Pore Complexes

Nuclear pore complexes (NPCs) allow selective import and export while forming a barrier for untargeted proteins. Using fluorescence microscopy, we measured in vivo the permeability of the Saccharomyces cerevisiae NPC for multidomain proteins of different sizes and found that soluble proteins of 150 kDa and membrane proteins with an extralumenal domain of 90 kDa were still partly localized in the nucleus on a time scale of hours. The NPCs thus form only a weak barrier for the majority of yeast proteins, given their monomeric size. Using FGΔ-mutant strains, we showed that specific combinations of Nups, especially with Nup100, but not the total mass of FG-nups per pore, were important for forming the barrier. Models of the disordered phase of wild-type and mutant NPCs were generated using a one bead per amino acid molecular dynamics model. The permeability measurements correlated with the density predictions from coarse-grained molecular dynamics simulations in the center of the NPC. The combined in vivo and computational approach provides a framework for elucidating the structural and functional properties of the permeability barrier of nuclear pore complexes.

scholarly journals The Effect of FG-Nup Phosphorylation on NPC Selectivity: A One-Bead-Per-Amino-Acid Molecular Dynamics Study

2019 ◽  
Vol 20 (3) ◽  
pp. 596 ◽  
Author(s):  
Ankur Mishra ◽  
Wouter Sipma ◽  
Liesbeth Veenhoff ◽  
Erik Van der Giessen ◽  
Patrick Onck
Keyword(s):  
Molecular Dynamics ◽  
Amino Acid ◽  
Intrinsically Disordered Proteins ◽  
Protein Complexes ◽  
Disordered Proteins ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Intrinsically Disordered ◽  
Dynamics Simulations ◽  
Pore Complexes

Nuclear pore complexes (NPCs) are large protein complexes embedded in the nuclear envelope separating the cytoplasm from the nucleoplasm in eukaryotic cells. They function as selective gates for the transport of molecules in and out of the nucleus. The inner wall of the NPC is coated with intrinsically disordered proteins rich in phenylalanine-glycine repeats (FG-repeats), which are responsible for the intriguing selectivity of NPCs. The phosphorylation state of the FG-Nups is controlled by kinases and phosphatases. In the current study, we extended our one-bead-per-amino-acid (1BPA) model for intrinsically disordered proteins to account for phosphorylation. With this, we performed molecular dynamics simulations to probe the effect of phosphorylation on the Stokes radius of isolated FG-Nups, and on the structure and transport properties of the NPC. Our results indicate that phosphorylation causes a reduced attraction between the residues, leading to an extension of the FG-Nups and the formation of a significantly less dense FG-network inside the NPC. Furthermore, our simulations show that upon phosphorylation, the transport rate of inert molecules increases, while that of nuclear transport receptors decreases, which can be rationalized in terms of modified hydrophobic, electrostatic, and steric interactions. Altogether, our models provide a molecular framework to explain how extensive phosphorylation of FG-Nups decreases the selectivity of the NPC.

scholarly journals Nanoscale electrostatic gating of molecular transport through nuclear pore complexes as probed by scanning electrochemical microscopy

2019 ◽  
Vol 10 (34) ◽  
pp. 7929-7936
Author(s):  
Pavithra Pathirathna ◽  
Ryan J. Balla ◽  
Guanqun Meng ◽  
Zemeng Wei ◽  
Shigeru Amemiya
Keyword(s):  
Amino Acids ◽  
Nuclear Pore Complex ◽  
Molecular Transport ◽  
Scanning Electrochemical Microscopy ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Electrostatic Gating ◽  
Pore Complexes ◽  
Electrochemical Microscopy

The nuclear pore complex (NPC) uses positive residues of amino acids to electrostatically regulate molecular transport through the peripheral route.

scholarly journals Percolation transition determines protein size limit for passive transport through the nuclear pore complex

2021 ◽  
Author(s):  
David N Winogradoff ◽  
Han-Yi Chou ◽  
Christopher Maffeo ◽  
Aleksei Aksimentiev
Keyword(s):  
Theoretical Model ◽  
Molecular Mechanisms ◽  
Coarse Grained ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Diffusion ◽  
Passive Transport ◽  
Microscopic Mechanism ◽  
Protein Size ◽  
Pore Complexes

Nuclear pore complexes (NPCs) control biomolecular transport in and out of the nucleus. Disordered nucleoporins in the complex's central pore form a permeation barrier, preventing unassisted transport of large biomolecules. Here, we combine coarse-grained simulations of an experimentally-derived NPC structure with a theoretical model to determine the microscopic mechanism of passive transport. Brute-force simulations of protein diffusion through the NPC reveal telegraph-like behavior, where prolonged diffusion on one side of the NPC is interrupted by rapid crossings to the other. We rationalize this behavior using a theoretical model that reproduces the energetics and kinetics of permeation solely from statistical analysis of transient voids within the disordered mesh. As the protein size increases, the mesh transforms from a soft to a hard barrier, enabling orders-of-magnitude reduction in permeation rate for proteins beyond the percolation size threshold. Our model enables exploration of alternative NPC architectures and sets the stage for uncovering molecular mechanisms of facilitated nuclear transport.

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

2010 ◽  
Vol 391 (7) ◽  
Author(s):  
Orit Peleg ◽  
Roderick Y.H. Lim
Keyword(s):  
Nuclear Pore Complex ◽  
Intrinsically Disordered Proteins ◽  
Molecular Transport ◽  
Disordered Proteins ◽  
Nuclear Pore ◽  
Biological Mechanisms ◽  
Intrinsically Disordered ◽  
Polymeric Chains ◽  
Insight Into

Abstract 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 beyond.

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