Simulations of nuclear pore transport yield mechanistic insights and quantitative predictions

Abstract

To study transport through the nuclear pore complex, we developed a computational simulation that is based on known structural elements rather than a particular transport model. Results agree with a variety of experimental data including size cutoff for cargo transport with (30-nm diameter) and without (<10 nm) nuclear localization signals (NLS), macroscopic transport rates (hundreds per second), and single cargo transit times (milliseconds). The recently observed bimodal cargo distribution is predicted, as is the relative invariance of single cargo transit times out to large size (even as macroscopic transport rate decreases). Additional predictions concern the effects of the number of NLS tags, the RanGTP gradient, and phenylalanine-glycine nucleopore protein (FG-Nup) structure, flexibility, and cross-linking. Results are consistent with and elucidate the molecular mechanisms of some existing hypotheses (selective phase, virtual gate, and selective gate models). A model emerges that is a hybrid of a number of preexisting models as well as a Brownian ratchet model, in which a cargo-karyopherin complex remains bound to the same FG-Nups for its entire trajectory through the nuclear pore complex until RanGTP severs the cargo-Nup bonds to effect release into the nucleus. mathematical modeling | molecular motor | nuclear-cytoplasmic transport | nucleoporins | filament dynamics S ignificant advances in our understanding of the nuclear pore complex (NPC), which mediates all transport between nucleus and cytoplasm, include a cataloging of the structural components, characterization of the transport factors, assays for rates of transport, including measurements of single molecule transit, some preliminary reconstitutions of nuclear transport, structural studies both at the cryo-EM and the X-ray crystallographic level, and molecular dynamics simulations between select components (1).Qualitative models to explain the selectivity of NPC transport for specifically tagged [nuclear localization signal (NLS)] cargo focus on the roles of the soluble factors and structural components of the pore. Two main soluble factors are Ran and the karyopherins ("kaps," also known as exportins or importins). The kaps are transport receptors that bind with high affinity to NLS cargo, whereas Ran is a small GTPase that exists in a gradient of its GTP:GDP form from the nucleus to cytoplasm and is involved in cargo release. The structural components are flexible filamentous phenylalanine-glycine nucleopore proteins (FG-Nups) that fill the central core of the pore. They are considered relatively "unstructured"-in vitro they lack secondary structure-and they have a series of repeats of the amino acid motif FG, varying from 6 to 43 per filament (2) and of various forms such as FxFG, GLFG, PSFG, or xxFG. All of the FG-Nups are arranged in eightfold symmetry, with some as a single set and some as two or four rings. Although the FG-Nups are essential for selective transport through the nuclear pore, many are dispensable. In yeast, up to 50% of the FG...

Effect of charge, hydrophobicity, and sequence of nucleoporins on the translocation of model particles through the nuclear pore complex

“…Even so, our model of the FG-Nups is coarsegrained in the sense that it makes no distinction between the hydrophobicities of the different amino acids, does not explicitly incorporate hydrogen bonding, and does not include specific interchain binding [e.g., as observed in the formation of gels with Asn-rich FG sequences (35)]. We have also omitted specific binding interactions between the model particles and the FG domains in the FG-Nups; these interactions have been proposed to play a role in the kap-mediated translocation mechanism (11,21,23,36,37). The association between the kaps and the FG domains is weak (36,38) and dynamical (22); therefore, the nature of this interaction is probably between high-affinity ligandreceptor binding and the hydrophobic interaction modeled here.…”

“…The kap-cargo complex particle interacts with the FG residues in this layer as it diffuses through the channel. Another simulation study suggested that the translocating particle remains bound to the same Nup for its entire trajectory through the NPC (21). The differences between these works arise due to the choice of the molecular model, which, in neither case, considered the specific sequence and length of the FG-Nups and the particular properties of each amino acid in the sequence (e.g., hydrophobicity, charge).…”

“…A definitive transport mechanism remains elusive because directly visualizing FG-Nups and the kap-cargo complex within individual NPCs is at the limits of current single-molecule tracking technology (15)(16)(17); therefore, theory (18,19) and computer simulations (20)(21)(22) have been used in an attempt to elucidate the essential features of the translocation process. In a recent coarsegrained molecular dynamics (MD) simulation, the kap-FG interaction was found to be highly dynamic and the FG-Nups formed a layer on the pore walls (20).…”

Molecular dynamics simulations of large macromolecular complexes

“…Another large protein-membrane assembly at the sub-cellular scale is the nuclear pore complex (NPC) [94, 95], located in the double bilayers of the nuclear envelope mediating particle exchange between nucleus and cytoplasm. Nsp1, a nuclear pore protein, is the major component of NPC.…”

Cooperative Interactions between Different Classes of Disordered Proteins Play a Functional Role in the Nuclear Pore Complex of Baker’s Yeast

“…Another model, the Di-block Copolymer Brush Gate (DCBG) model [11], Fig 1A, assumes that the key FG nups which regulate transport are individually bi-phasic, while most theoretical and polymer physics approaches [19–22] to the NPC transport problem tend to assume a homogenous structure for individual FG nups, resulting in a relatively homogenous NPC architecture. More recent advances in coarse grained simulations of the nucleoporins that consider sequence have alleviated this issue [23–29] but still lack the level of detail necessary to produce potential secondary or tertiary structure. More importantly, none of the theory or simulations to date have really shed light on the specific roles of the different types of FG nups within a single NPC and the functional reason for their co-existence.…”