Assembly of Nsp1 Nucleoporins Provides Insight into Nuclear Pore Complex Gating
[['Nuclear pore complexes ( NPCs ) are large protein assemblies embedded in the nuclear membrane that provide the only conduit for exchange of molecules between cytoplasm and nucleoplasm , a process defined as nucleocytoplasmic transport ( NCT ) .', 'While the membrane bound nucleus of a eukaryotic cell protectively envelopes the genetic material , separating it from the cytoplasm , processes such as DNA transcription require access to the genetic material in the interior of the nucleus by a myriad of molecules , including large molecules such as proteins .', 'Two modes exist for the NCT: passive diffusion of molecules , smaller than 40 kDa or 9–10 nm in diameter [1] , and selective transport into and out of the nucleus for larger molecules such as proteins , RNA , ribosomal subunits , the larger molecules being recognized by transport factors ( TFs ) , carrier proteins , which shuttle back and forth ferrying their cargo through the NPC .', 'NPCs are giant complexes having molecular masses ranging from ∼65 MDa ( in yeast ) to ∼125 MDa ( in higher eukaryotes ) that are made of copies of 30 different proteins termed nucleoporins ( nups ) [2]–[5] .', 'Recently , a detailed 3D model for the position and abundance of each nup in the Saccharomyces ( S . ) cerevisiae NPC structure was proposed based on experimental data obtained from molecular , biochemical and structural studies of the NPCs and their components [6] , [7] .', 'In the modeled structure , the scaffold of the NPC is formed by two protein subcomplexes that , through linker proteins , anchor a set of FG-containing nups [6] .', 'The complex has 8-fold symmetry about its central axis and 2-fold symmetry about the equatorial plane such that each nup is repeated 8- , 16- , 32- , or 48-fold .', 'Altogether , the NPC forms a multi-protein complex of nearly ∼450 proteins [4]–[7] .', 'About one-third of all pore proteins constitute the barrier proteins also known as FG-nups ( F and G represent amino acids phenylalanine and glycine , respectively ) .', 'FG-nups are intrinsically disordered , rich in FG-repeat motifs [4] , [5] , [8]–[10] and form the central transport channel of the NPC extending , however , also into the cytoplasmic and nucleoplasmic space .', 'While the FG-nups extending toward cytoplasm and nucleoplasm sides are asymmetric in distribution , the FG-nups ( eg . , Nsp1 ) of the central region have copies distributed symmetrically around the scaffold of the NPC and are critical for bidirectional NCT .', 'The FG-nups are present in different lengths ( a few to several hundred amino acids long ) and exhibit different amino acid composition and repeat motifs , typically FG , GLFG , or FxFG motifs ( x being any amino acid , largely serine ) separated by linker regions of 10–20 hydrophilic amino acids [4] , [9] , [10]; their different spatial localizations render the central channel heterogeneous .', 'These nups play a central role in selective transport of the NPC and this selectivity is attained by bearing specific binding sites ( the FG motifs ) for the TFs that undergo multiple , low-affinity interactions with nups as they permeate their way through the channel [11]–[17] .', 'Given that the central transport channel is filled with heterogeneous and natively unstructured FG-nups that are not susceptible to crystallization for structure determination , the important question how the unstructured FG nups are assembled in the interior to promote efficient and selective gating could not be answered unequivocally .', 'Several models have been proposed so far to explain the structure of the interior of the NPC .', 'According to the virtual gate model [4] , [18] , the tethered , unstructured FG-repeat proteins form an entropic barrier that repels non-specific cargo , preventing it from reaching the interior of the channel , whereas TFs that interact with the FG-repeats of nups in the NPC interior channel have higher probability of entering and permeating the channel .', 'An alternative model , namely the polymer brush model [19] , [20] , is based on the above model suggesting that the FG-nups form extended brush-like polymers with inter-linked bristles that reversibly collapse upon binding of TFs .', "TFs pass the channel by repetitive binding and unbinding to the nups' FG repeats until they reach the exit of the pore [21] .", 'In contrast , the selective phase/hydrogel model [22] proposes that FG nups in the central channel achieve a sieve-like meshwork through weak inter-repeat FG-FG hydrophobic interactions to form a hydrogel within the central channel [23] .', 'TFs bind to the FG-repeats via low-affinity interactions which transiently melt these FG-FG cross-links of the meshwork , allowing thus only TFs to permeate the channel .', 'Furthermore , non-specific cargo that cannot bind is filtered out [23] , [24] .', 'Another model , the reduction of dimensionality ( ROD ) model , proposes that the TFs act as ferries , carrying cargo and sliding on the surface of phenylalanine-glycine ( FG ) motifs by interacting with the FGs according to a 2D random walk rather than a 3D diffusion [25] .', 'The ‘forest’ model , later modified to the tube gate model [26] , [27] , proposes the interior of the NPC to be a hydrogel and the periphery to be brush-like , featuring two separate zones of traffic with distinct physiochemical properties .', 'In their study , based on the hydrodynamic radius of individual nups localized in different regions of the NPC , the respective authors classify nups as', '( a ) ‘shrubs’ that form cohesive domains with collapsed-coil and low charge content;', '( b ) ‘extended coils’ that form high-charge content , non-cohesive domains;', '( c ) ‘trees’ having features of both', '( a ) and', '( b ) .', 'Thus , a ‘forest-like’ structure of FG-nups is formed in the NPC .', 'In this model , the collapsed-coil domains are hypothesized to form a transport zone 1 in the central pore and the extended-coil domains form a peripheral zone 2 .', 'The forest model is based on the observations of individual nup conformations , not considering interactions between nups .', 'In a recent study on the role of nups in NPC gating , Tagliazucchi et al [28] deduced a potential of mean force of TF transport from the amino acid ( positive , negative , hydrophobic ) distribution and claimed that this potential can explain the use of TFs for traffic across the nuclear envelope .', 'The claim is based , however , on the assumption that nups are individually random and do not form any structures in the channel interior as suggested by the earlier models of the NPC interior discussed above .', 'The authors also do not attribute an explicit role to TFs interacting with nups through FG motifs .', 'Most models propose an FG network as the main mechanism for pore selectivity and address to a much smaller degree a role for the actual molecular structures formed by the disordered nups of the NPC .', 'Likely , the qualitative differences of the models can be reconciled with the heterogeneity found along entire transport pathways through the NPC that furnishes different environments on the cytoplasmic side , in the interior and on the nucleoplasm side of the NPC .', 'To identify computationally the structure formed by the nups in the interior of the NPC pore , Miao and Schulten [20] carried out molecular dynamics ( MD ) simulations of nup polymers , using coarse-grained MD .', 'Unlike the other approaches arriving at models for NPC gating , their approach included details of individual amino acids and , more importantly , included the interactions between nup polymer chains .', 'In the simulations these chains were end-grafted to a flat surface in a 5×5 array .', 'The MD simulations suggest that the nups have a strong tendency to form bundles of typically 2–6 proteins and that the bundles are inter-linked in a mesh-like manner as single nups cross frequently from one bundle to another one .', 'From this behavior results a brush structure that is made of bundles of interlinked bristles .', 'This structure can explain to some degree the gating of NPC pores: the structure can be passed by small biomolecules readily while larger biomolecules need to alter the structure to pass through .', 'This role , namely of cutting through the mesh-like structure , could be played by transport factors .', 'While the structure model of Miao and Schulten is highly suggestive , the study had obvious shortcomings imposed at the time by limitations in available computer power .', 'One shortcoming is the short length ( 100-aa ) of nups employed that is in contrast to the actual , much longer ( about 600-aa ) lengths of many nups .', 'A second shortcoming is that only a single type of nup tethering was investigated , namely tethering to a flat surface .', 'A third shortcoming is that the simulated ensemble started with all nups initially in a highly ordered , namely fully-extended , straight chainconformation .', 'Enjoying today in the form of petascale computers tremendously more computer power than was available to Miao and Schulten we have performed for the present study microsecond-long simulations on various conformational ensembles of the 609-aa-long FG domain of Nsp1 ( hence termed Nsp1-FG ) , a key nup in the NPC central channel .', 'Three different ensembles were investigated , namely ensembles of untethered Nsp1-FGs , of Nsp1-FGs tethered to a flat surface as in the Miao and Schulten study , and of Nsp1-FGs tethered to a ring-like surface that is qualitatively similar to the geometry of the NPC .', 'Most essential , however , is that the present study explores Nsp1-FG ensembles in different initial protein conformations , namely fully-extended conformations as assumed by Miao and Schulten as well as random polymer-like conformations .', 'Nevertheless , the new MD simulations support the structural model as seen in the simulations of Miao and Schulten , in which 100-aa-long fragments of Nsp1-FG form bundles linked through single proteins crossing between bundles , but reveals more physical characteristics and a high degree of heterogeneity of the model .', "For example , the new and more extensive simulations demonstrate that the actual cross-linked bundle system depends to a significant degree on the actual tethering of nups' ends as well as on the initial nups conformation .", 'A further key finding is that the FG motifs distributed in the cross-linking regions have highly accessible side groups that furnish potential binding epitopes for TFs to assist in cargo transport .'], ['To elucidate how the NPC poses a barrier for transport of molecules into and out of the nucleus , we thought to characterize through simulations the general structural features in the NPC central channel , arising from the assembly of disordered proteins .', 'We investigated the assembly using only one type of FG-nups , namely Nsp1 .', 'Nsp1 is an FxFG-rich nup present in the central channel and has been investigated as a model protein in previous studies [20] , [24] , [26] .', 'The nup is present in 32 copies in both the upper and lower half of the NPC equatorial plane; its location is ∼30 nm from the center of the NPC [29] .', 'The large number of copies and its spatial location inside the central channel renders Nsp1 critical for bi-directional nucleo-cytoplasmic transport ( NCT ) .', 'The 609-aa-long FG-domain of this protein , namely Nsp1-FG , was described through coarse-grained simulations that investigated the structures formed by different ensembles of Nsp1-FGs .', 'In two first simulations , the proteins were tethered to a gold ring surface ( simulations wild-type_ring and mutant_ring ) .', 'For the initial protein conformations we assumed fully-extended Nsp1-FGs as also adopted in the earlier study by Miao et al [20] .', 'However , rather than a 100-aa-long fragment of FG-domain simulated in their study , we used in the present study the full-length ( 609-aa-long ) FG-domain .', 'The two simulations ( wild-type_ring and mutant_ring ) differ in that the first employs wild-type Nsp1-FG , whereas the second employs a mutant Nsp1-FG , with all its phenylalanines and glycines replaced by alanines .', 'In a third simulation , Nsp1-FGs were tethered to a flat gold surface ( simulation random_array ) .', 'In this case the proteins were assumed initially in a random polymer conformation .', 'In a fourth simulation the effect of end-tethering was investigated by simulating Nsp1-FGs untethered and initially homogeneously distributed in bulk solvent in random polymer conformations ( simulation random_bath ) .', 'All ensembles contain the protein at a concentration that is characteristic for the interior of the NPC channel .', 'The simulations carried out are listed in Table', '1 . For simulation wild-type_ring , Nsp1-FGs were grafted to a gold nano-ring that matches the pore dimension of the pore system designed by an experimental group [21] , [29] , engineered to mimic the NPC geometry .', 'The system was simulated for after which time the initially fully stretched Nsp1-FGs formed brush-like bundle structures ( see Figure 1 ) , the same structure as reported in an earlier study of 100-aa-long fragments of FG-domains [20] .', 'Such structures can potentially function as a selective barrier to transport , supporting the virtual-gate model of nuclear pore gating [1] , [20] .', 'Key for the gating mechanism is that the bundles are interconnected via single Nsp1-FG chains cross-linking adjacent bundles ( see Figure 1 ( c ) and Figure 1 ( e ) ) .', 'We analyzed the observed brush-like bundle structures through computation of the bundle-thickness distribution .', 'Bundle thickness is determined in terms of the number of different Nsp1-FG chains present in a bundle .', 'For the brush-like structure as shown in Figure 1 , bundles with few chains are most favored entropically , yet the bundle thickness distribution shows that there arise also thick bundles that consist of more than fourteen chains ( Figure 2 ) .', 'To test which role FG-repeats play in the formation of the structure shown in Figure 1 , we simulated a mutant system replacing all phenylalanines and glycines in Nsp1-FG by alanines ( simulation mutant_ring ) .', 'The assembly structure of mutant Nsp1-FGs in the nanopore model resulting from simulations is very similar to that seen for wild-type ( WT ) Nsp1-FGs .', 'The similarity is also reflected in the bundle distribution shown in Figure 2 that shows a bimodal shape with thin and thick mutant Nsp1-FG bundles arising .', 'The dynamics of Nsp1-FG assembly during the simulation and a detailed 360-degree view of the brush-like structures that resulted from the simulation are provided in Videos S1 and S2 , respectively .', 'The initially fully- extended and straight-chain Nsp1-FGs , simulated in [20] and in the present simulations ( wild-type_ring and mutant_ring ) , are highly ordered at the outset .', 'These simulations may lead to trapped and , thus , unrealistic states that are in quasi-equilibrium , calling into question if the assembly structure shown in Figure 1 is representative for the NPC interior .', 'Therefore , we modeled in simulation random_array end-tethered Nsp1-FGs with a grid spacing of 6 nm , similar to that in the gold ring geometry , but assumed , for the initial state , random rather than straight ( Nsp1-FG ) conformations .', 'The simulation involved again the full-length ( 609-aa ) Nsp1 FG-domain , Nsp1-FG .', 'From the present simulation resulted , nevertheless , a brush-like structure of bundles with the bundle distribution shown in Figure', '2 . However , the bundles formed exhibit more cross-links than seen in the earlier simulation [20] ( see Figure 3 ) .', 'A view of the formation , during simulation random_array , of brush-like structures with cross-linked bristles and a 360-degree view of the final conformation are shown in Videos S3 and S4 , respectively .', 'The brush-like structure resulting for ensembles of closely spaced Nsp1-FGs can similarly be characterized by brush height .', 'For the end-tethered Nsp1-FGs in simulations wild-type_ring , mutant_ring and random_array we monitored the average brush height , namely the end-to-end distance of the main-chain Cα beads of the Nsp1-FG chains in the -direction ( Figure 4 ) .', 'The average was taken over all Nsp1-FG chains in the given simulation system .', 'The Nsp1-FG chains in the final structures for both simulations , namely wild-type_ring and mutant_ring , exhibit similar average height of about 80 nm .', 'Interestingly , we observed that in both cases the average height decayed over time in a similar fashion on the time-scales of the simulations .', 'The Nsp1-FG chains in the final structure of the simulation random_array exhibited an average height of 60 nm .', 'In order to investigate how the final structure of closely spaced Nsp1-FGs is affected by end-tethering , we carried out a simulation of initially fully disordered Nsp1-FGs freely floating in a bath ( random_bath ) at a protein density similar to that in random_array .', 'These Nsp1-FGs formed again bundles ( see Figure 5 ) , but in this case with many more links between bundles ( formed by single Nsp1-FGs crossing between bundles ) than seen in case of simulations wild-type_ring , mutant_ring and random_array .', 'The resulting mesh-like structure can be clearly distinguished from the brush-like structures of wild-type_ring , mutant_ring and random_array through the bundle thickness distribution shown in Figure 2 as the bundles arising in simulation random_bath exhibit at most fifteen chains per bundle .', 'Videos S5 and S6 show the dynamics of Nsp1-FGs as seen in simulation random_bath as well as a 360-degree view of the resulting structure , respectively .', 'Given the different structures formed by Nsp1-FG assemblies , as they result from the present simulations and represent likely the interior of the NPC , one wonders if any specific interactions , in particular , hydrophobic FG-FG interactions , favor the structures seen .', 'We determined , therefore , for the different types of amino acids involved in the formation of bundles how often particular amino acids arise in bundles .', 'For simulation wild-type_ring all types of amino acids as well as FG motifs exhibit a similar propensity to be involved in bundle formation , suggesting that FGs are not particularly critical for the formation of these structures ( Figure S1 ) .', 'This is also supported by simulation mutant_ring , in which the FG motif was replaced by alanines , and by an earlier study [20] .', 'Moreover , all amino acids , including both hydrophilic and hydrophobic residues , are equally favorable for the brush-like structures of the wild-type_ring .', 'Actually , no structures arising from simulations wild-type_ring , mutant_ring , random_array or random_bath exhibit particular amino acid preferences .', 'Apparently , formation of the observed bundle structures is not sequence-specific and likely comes about through contributions from both hydrophobic and hydrophilic interactions .', 'However , Dolker et al [30] showed hydrophilic residues in the linker regions to play a key role in the nup aggregation process and structure , whereas the role of aromatic phenylalanine residues is less dominant .', 'To further characterize the structural details of bundles , we performed a so-called reverse CG calculation [31]–[33] on a fragment comprising cross-linked bundles arising in the final structure of simulation wild-type_ring .', 'Such calculation assigns an all-atom ( AA ) model consistent with a CG model .', 'We then employed this AA model as a starting point of a 100-ns equilibration simulation ( simulation fragment_AA ) .', 'These fragments of bundles increased bundle thickness during the equilibration ( see Figure S2 ) , likely due to the absence of end-tethering of proteins resulting from the fragmentation applied .', 'Inter-chain backbone-backbone hydrogen bonds ( HBs ) frequently formed ( ∼30–50% ) for most amino acids within the bundles , and played a critical role for the stability of the bundles ( Figure 6a ) .', 'Secondary structure analysis , on the other hand , revealed no apparent preference ( <5% ) for either α-helix or β-sheet formation for any amino acid .', 'To assess the role of FG and FxFG motifs on nup assembly , we determined the solvent accessible surface area ( SASA ) for individual amino acids; the results are shown in Figure 6b for the FG and FxFG motifs present in a bundle region or in a cross-linking region .', 'The SASA values from both CG and AA simulations show that both FxFGs and FGs adopt a higher degree of solvent accessibility when present in a cross-linking region than compared to SASA values arising for these motifs in a bundle region ( Figure 6b ) ; in this respect FxFG and FG motifs behave very similarly ( Figure 6b ) .', 'In order to determine in how far the structures of Nsp1-FG assemblies described above provide a barrier against the diffusion of molecules through them , we computed the size of passing molecules as described in Methods and illustrated in Figure 7 .', 'Table 2 lists for the structures resulting from simulations wild-type_ring , mutant_ring , random_array , and random_bath the average radius for the cargo molecules that can diffuse through the structures .', 'Large pores are available for diffusive passage when Nsp1-FGs are assembled into bundle structures with few cross-links; in this case molecules with a radius as large as ∼77 Å can pass , whereas mesh-like bundle structures with many cross-links furnish only relatively small pores for molecules to pass through , namely only ones with radii smaller than 43–50 Å .', 'Our results suggest structural properties of closely interacting Nsp1-FG proteins .', 'We contrast these properties with the structural property of an isolated Nsp1-FG protein .', 'The structures exhibited by a single untethered Nsp1-FG protein monitored in a CG MD simulation showed very different features from the structures resulting from the ensembles of closely spaced Nsp1-FG proteins in simulations wild-type_ring , mutant_ring and random_array .', 'An initially fully-extended , untethered single Nsp1-FG is found to coil up into a globule-like structure , with its radius of gyration ( Rg ) decreasing from over 300 Å ( corresponding to the initially fully-extended form ) to 65 Å ( corresponding to the final , coiled form ) as shown in Figure S3 .', 'Similar globule-like structures were also observed in earlier studies of dynamics of individual , end-tethered short fragments of Nsp1-FGs [20] .'], ['The structure of the NPC central channel interior , made up of intrinsically disordered nups with FG-repeats , governs transport through the NPC .', 'The structure formed by the assembly of tethered nups determines the size of small molecules that can passively diffuse through .', 'The structure also interacts , likely through the FG repeats , with the transport factors that carry various cargoes through the NPC .', 'To characterize the structure of the channel interior , we investigated the assembly using one type of FG-nups , namely Nsp1-FG , that is present in the central channel .', 'We studied , using CG MD simulations , the dynamics of Nsp1-FGs starting from three different initial states: tethered , straight-chain conformations; tethered , random-chain conformations; and untethered , random-chain conformations , simulating each system for 1 µs .', 'We observed in all cases the formation of brush-like bundles linked through single Nsp1-FGs crossing between bundles .', 'This assembly structure is very similar to one seen in prior simulations of initially fully-extended , Nsp1-FG fragments ( 100-aa-long ) , that were closely grafted ( 2 . 6 nm apart compared to 6 nm-spacing in the present study ) in an array-like arrangement [20] .', 'On the other hand , a new finding of the current study is that frequency of crossings between bundles and bundle thickness depend on protein length , on the geometry of the simulated volume , on the degree of tethering , and , particularly , on the initial conformations of the proteins .', 'An interesting observation is that FGs may not be the only factor that plays a role in the formation of bundles .', 'In our present mutant study ( simulation mutant_ring ) and that reported in [20] , basically the same structures of brush-like bundles with cross-linking nups are formed as with wild-type Nsp1-FGs , suggesting that factors other than FG motifs could also contribute to the formation of bundle structures .', 'This finding is also supported by our analysis showing that all kinds of amino acids have similar propensity to arise in the bundles formed .', 'This finding is in contrast to a study of cross-linking arising in a hydrogel-like structure suggested for the selective phase model: in such structure low affinity inter-FG hydrophobic interactions , supposedly , are responsible for cross-linking [23] , [24] .', 'We also note that Miller et al [34] showed through EM experiments that another nup , namely Nup153 , is less aggregation-prone when having its FG motifs either deleted or mutated into AG .', 'However , owing to different nups and mutations investigated in their study and ours , we are unable to address the discrepancy between the two studies .', 'The FG motifs not only play a role in bundle formation but also assist in transport of TF through the NPC channel .', 'A recent study showed that the presence of unbound and , thus , freely available FG-repeat motifs helps binding all possible sites of the TF and is required for efficient cargo transport [35] .', 'In line with that study , a portion of the FxFG and FG motifs are found in the present study , through a SASA analysis in the framework of both CG and all-atom simulations , to be highly accessible to solvent in the cross-linking regions between the bundles .', 'Accordingly TFs , dotted with FG binding sites , should bind to the multiple FGs arising in the cross-linking regions and , perhaps , tear the bundles apart in a zipper-like fashion .', 'Our interpretation , based on the structural features observed for tethered , intrinsically disordered FG-nups of the transport channel , agrees with the other views [18]–[21] , [23]–[27] , [36] in that the nups form characteristic quasi-stable , i . e . , slowly varying , structures bearing FG motifs , with which TFs are known to interact .', 'Thus , interaction between TF surfaces and nups is structured and cannot solely be described properly by employing the potential of mean force , as suggested by Tagliazucchi et .', 'al , which does not include the assembly of nups in the interior of the channel [28] .', 'Experiments employing single-molecule studies that deduced spatial density plots for TF-FG-repeat interactions have also shown that the interaction sites are not evenly distributed in the NPC; instead , they form spatial clusters inside and outside the central nuclear pore [37] .', 'Other models have been suggested for the structures formed by nups in the NPC .', 'For example , experiment and simulation suggest that nups aggregate as amyloid like fibers , in which β-sheet rich sheets form through inter-chain backbone-backbone hydrogen bonding and then through stacking of these sheets .', 'Indeed solid-state NMR data are consistent with that such amyloid-like structures in gel-like aggregates formed by Nsp1-FGs mainly through interactions between hydrophilic residues [38] .', 'Likewise , all-atom simulations of ensembles involving the small peptide FSFG showed that the hydrophobic residue phenylalanine can form amyloid-like aggregates [30] , a feature apparently also consistent with a high-resolution EM study of nup aggregates [34] .', 'While the MARTINI force field [39] employed in the present study is unable to model β-sheets , our all-atom simulations based on a reverse CG calculation as described above is capable of yielding β-sheet formation , and indeed resulted in considerable inter-chain backbone-backbone hydrogen bonds in the bundles , but yielded only a small degree of β-sheet structure .', 'Clearly , the structural variety of actual nups aggregates in the NPC still needs to be investigated further through experimental and computational means .', 'A recent model [27] for the structure inside a NPC transport channel proposed that the central region is gel-like and the periphery is brush-like .', 'The authors postulate two zones , a central and a peripheral zone , for trafficking of molecules in and out of the NPC .', 'Similarly , we model the structure of the entire transport channel of the NPC based on our simulation results .', 'In our study , we did not simulate the entire region of the channel interior as this is computationally still too expensive presently .', 'Instead , we simulated systems of Nsp1-FGs starting from different initial states and based on the results suggests a heterogenous model for the NPC central channel:', '( i ) at the periphery , i . e . , close to the scaffold , the FG nups are end-tethered to the scaffold through linker proteins;', '( ii ) in the middle region , FG nups adopt natively disordered conformations filling up the central volume .', 'The salient features of nups in a real NPC channel arise in our model through end-tethering of nups , protein density , random conformations for initial states , and a full-length real FG-nup found in the NPC , namely the Nsp1-FG domain .', 'Therefore , we interpret our simulation results then to imply that the nups form different structures in different regions of the central channel ( see Figure 8 ) .', 'The region in the periphery of the central channel , away from the center , should be represented by a system of tethered nups as in simulation random_array , where brush-like bundles with less cross-linking arise .', 'The NPC inner diameter is about 30 nm and unstructured nups that are several hundred amino acids long span this volume of an open pore .', 'Since tethering effects should be minimal in the central region due to the large distance from the NPC wall , where nups are actually tethered , we expect that the structure found in this region is similar to that seen in simulation random_bath , exhibiting bundles with a high degree of cross-linking forming a mesh .', 'In this region , we observed the pore size to be 9–10 nm ( Table 2 ) which agrees with the experimentally determined size limit for passive diffusion of small molecules .', 'In the present study we modeled a homogeneous system of nups consisting of only Nsp1-FGs .', 'FG-nups in the real NPC central channel are heterogeneous; FG-nups vary across the channel volume in length , amino acid composition , FG repeat motifs ( FG , FxFG and GLFG ) , composition and spacing of the linker regions .', 'However , both simulation of a short , 100-aa-long fragment of Nsp1-FG in [20] and the current simulation of a full length Nsp1-FG ( 609-aa-long ) led to the same brush-like structure model , suggesting that the interlinked brush-like structure discovered are not sensitive to varying length and type of proteins .', 'Also , nups are known to be intrinsically disordered , interacting with each other through FG-repeats distributed throughout their entire sequences .', 'Such disorder and interactions between FG-repeats were also captured in the present model .', 'Therefore , we suggest that FG nup heterogeneity does not yield qualitatively different nup assembly structures .', 'In addition to the structural features of the NPC , it is necessary for an understanding of the transport mechanism of the NPC to examine the dynamics of assembly and conformational change of the nup proteins .', 'Transport arises on a millisecond timescale [40] , which is beyond the microsecond time scale accessible to large scale MD simulation and performed in the present study .', 'Thus , we are unable to infer information of dynamics of the transport process from the current simulations .', 'Instead , our simulation results should be interpreted as quasi-static pictures of dynamic assembly structures of the nups .', 'The major unsolved problem regarding our still limited knowledge of the NPC and NCT is how specific large cargoes with the assistance of TFs manage to pass the NPC .', 'Clearly the NPC being one of the largest and at the same time one of the most dynamic macromolecular systems in eukaryotic cells still holds great secrets and offers opportunity for great discoveries .', 'Our suggestion here of the principal assembly structure of disordered nups in the NPC , even if completely true , does not imply yet how
['Introduction', 'Results', 'Discussion', 'Methods']
['Nuclear pore complexes ( NPCs ) form gateways for material transfer across the nuclear envelope of eukaryotic cells .', 'Disordered proteins , rich in phenylalanine-glycine repeat motifs ( FG-nups ) , form the central transport channel .', 'Understanding how nups are arranged in the interior of the NPC may explain how NPC functions as a selectivity filter for transport of large molecules and a sieve-like filter for diffusion of small molecules ( < or ) .', 'We employed molecular dynamics to model the structures formed by various assemblies of one kind of nup , namely the 609-aa-long FG domain of Nsp1 ( Nsp1-FG ) .', 'The simulations started from different initial conformations and geometrical arrangements of Nsp1-FGs .', 'In all cases Nsp1-FGs collectively formed brush-like structures with bristles made of bundles of 2–27 nups , however , the bundles being cross-linked through single nups leaving one bundle and joining a nearby one .', 'The degree of cross-linking varies with different initial nup conformations and arrangements .', 'Structural analysis reveals that FG-repeats of the nups not only involve formation of bundle structures , but are abundantly present in cross-linking regions where the epitopes of FG-repeats are highly accessible .', 'Large molecules that are assisted by transport factors ( TFs ) are selectively transported through NPC apparently by binding to FG-nups through populated FG-binding pockets on the TF surface .', 'Therefore , our finding suggests that TFs bind concertedly to multiple FGs in cross-linking regions and break-up the bundles to create wide pores for themselves and their cargoes to pass .', 'In addition , the cross-linking between Nsp1-FG bundles , arising from simulations , is found to set a molecular size limit of < for passive diffusion of molecules .', 'Our simulations suggest that the NPC central channel , near the periphery where tethering of nups is dominant , features brush-like moderately cross-linked bundles , but in the central region , where tethering loses its effect , features a sieve-like structure of bundles and frequent cross-links .']
['Cells of higher life forms separate their genomes from the rest of the cell in a nucleus that surrounds the genome by a nuclear envelope .', 'Hundreds of pores , each a complex made of many proteins , assure traffic into and out of the nucleus through highly selective transport: small biomolecules can pass unhindered , whereas large biomolecules need to associate with proteins called transport factors , to pass .', 'Little is known about how the nuclear pore complexes function , a key impediment to observation being their huge size and the disordered nature of the pore interior .', 'We investigated computationally what kind of structure the nuclear pore proteins ( nups ) form .', 'In the computation we place many nups , each a 600 amino acid-long protein , into arrangements considered representative for the nuclear pore , and simulate the subsequent molecular behavior .', 'We find that the nups form bundles of 2–27 proteins , the bundles being cross-linked when a single nup leaves a bundle and joins an adjacent one .', 'The finding suggests an adaptive molecular mesh arrangement of nups in the nuclear pore and explains how selective transport is accomplished , namely that passage of sufficiently small molecules is unhindered by the cross-linking , but that large molecules need the assistance of transport factors to melt the cross-linking .']
['biophysics', 'biology', 'computational biology']