Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Abstract

Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.nuclear pore | molecular dynamics | nucleoporins | transport factors | NMR T he nuclear pore complex (NPC) regulates macromolecular transport between the nucleus and the cytoplasm in all eukaryotic cells (1, 2). The NPC allows for passive diffusion of small molecules including sugars, ions, and water, while simultaneously imposing size-dependent exclusion of macromolecules without nuclear transport factor (TF) binding sites, generating unique nuclear and cytoplasmic compartments (3, 4). Larger macromolecules can bypass the selectivity barrier of the NPC by interacting with TFs that shuttle cargo through the central channel of the pore. In pathological conditions, including many cancers and viral infections, the NPC is hijacked and used to transport undesired particles, or it is modified to attenuate the cellular responses to disease onset (5, 6).TFs traverse the NPC by selective and reversible association with disordered phenylalanine-glycine (FG) repeat domains of the FG nucleoporin proteins (FG Nups), which line the surface of the NPC (7-14). Each FG repeat domain consists of 5-50 FG repeats. In turn, each FG repeat consists of a single FG motif of various sequence flavors, such as the FSFG and GLFG motifs, and 10-30 spacer residues that separate consecutive FG motifs (15, 16). The spacer residues are strongly hydrophilic (17) and rich ...

Thermodynamic characterization of the multivalent interactions underlying rapid and selective translocation through the nuclear pore complex

“…Our previous simulations demonstrated that FSFG motifs make multiple transient and exploratory contacts with the interaction surface of NTF2 (24). Taken together, the single binding mode detectable by SVD suggests that the exchange rates of FG motifs contacting the NTF2 surface are faster than the experimental detection (~ms) such that the observed CSPs reflect a single time-averaged binding mode.…”

Integrative structure and functional anatomy of a nuclear pore complex

“…Finally, the remaining regions without an atomic representation ( i.e. , the predicted transmembrane and disordered regions) were represented by a flexible string of beads encompassing 25 to 100 residues each; the low-resolution representation of these regions is justified because their conformations are likely “decoupled” from the structure of the rest of the NPC 48,118 .…”

“…Each of the FG repeat domains was represented as a flexible string of beads; a bead had a radius of 6 Å and encompassed 20 residues to achieve a compromise between computational efficiency and accuracy 118,146–149 . Consecutive beads were restrained by a bond with an equilibrium length of 18 Å and a constant force of 1.0 kcal/mol/Å, approximating the spring-like nature of flexible polymers 150 in general and FG repeat domains 118,147–149,151–153 in particular. The freely diffusing molecules included 1,600 NTRs and 1,600 inert macromolecules (0.33 mM each), each consisting of eight subgroups of 200 macromolecules, ranging in radius from 4 to 28 Å in 2 Å increments (10 to 75 kDa, assuming constant protein density of 1.38 g/cm 2 ).…”

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes

“…This technique has been combined with recent advances in MS instrument technology and complementary structural techniques, such as cryoelectron microscopy, to interrogate the structures of highly complex protein structures (Gaubitz et al, 2015; Greber et al, 2014; Lasker et al, 2012; Navare et al, 2015; Olson et al, 2014; Raveh et al, 2016). A seminal in silico study demonstrated that homology modeling could determine highly accurate structures when the modeling was combined with a sufficient density of distance constraints derived from chemical crosslinking (Leitner et al, 2010).…”