Ran modulates allosteric crosstalk between importin β surfaces

Domain: neuroscience
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
A cellular gradient of the GTPase Ran orchestrates the movement of import and export complexes through the Nuclear Pore Complex (NPC). Ran-GTP modulates two essential activities of importin β during nuclear import. On the one hand, it reduces the avidity of importin β for phenylalanine-glycine-rich nucleoporins (FG-nups), thereby facilitating the passage of import complexes through the permeability barrier. On the other hand, it disassembles import complexes, releasing the import cargo into the nucleus. The precise mechanisms by which Ran-GTP modulates importin β activities have remained hypothetical. Leveraging cryogenic electron microscopy (cryo-EM) single-particle analysis, in this paper, we describe five distinct conformational states of importin β in complex with various effectors encountered during an import reaction, specifically IBB-cargos, FG-repeats, Ran-GTP, Ran-GTP:RanBP1, and Ran-GDP:RanBP1. Comparing these states allows us to decipher the conformational landscape of importin β without interference from crystallization agents and lattice forces. By correlating structural data with biochemical activities, we find that Ran-GTP, but not Ran-GDP, constrains the solenoid structure of importin β, closing high-affinity FG-binding pockets and displacing import cargos through allosteric crosstalk between the concave and convex surfaces. We propose that this allosteric mechanism is relevant to other β-karyopherins involved in nuclear import.

Introduction
Importin β is the prototypical nuclear import receptor and the founding member of a family of transport receptors known as β-karyopherins. Importin β binds import cargos directly or through the adapter protein importin α, which exists in seven isoforms in humans. In the classical nuclear import pathway, cytoplasmic cargos containing a nuclear localization signal (NLS) assemble into a trimeric importin β:α1:NLS-cargo complex, which docks to the nuclear pore complex (NPC) and moves through the NPC channel due to importin β avidity for phenylalanine-glycine-rich nucleoporins (FG-nups). The chemical structure of the NPC inner channel is unknown, but ample evidence suggests FG-nups form a hydrogel that constitutes a selective permeability barrier. The small GTPase Ran, enriched as Ran-GTP in the nucleus and Ran-GDP in the cytoplasm, facilitates the import reaction by regulating the interaction between transport receptors (like importin β) and cargo, controlling the directionality of transport. Ran-GTP binds importin β with high affinity, releasing the NLS-cargo in the nucleus. It also decreases the affinity of importin β for FG-nups, preventing importin β from stalling and thus clogging the NPC. Importin β fragments that bind to the NPC but do not associate with Ran-GTP irreversibly clog the pore, inhibiting NLS-dependent protein import.

Importin β can shuttle through the NPC on its own, without cargo or an adapter. In digitonin-permeabilized cells, purified importin β accumulates at NPCs and can enter the nucleus independently of importin α or cargo. Single molecule/FRAP experiments directly observed importin β shuttling through NPCs, revealing transient interactions with FG repeats and a characteristic transport dwell time. A large portion of the entire cellular pool of importin β resides inside the NPC, possibly divided into two pools within the NPC: one pool is stably bound to the NPC, with a slow off-rate, regardless of the presence of Ran-GTP; the other pool is stably bound to the NPC only in the absence of Ran-GTP, which actively disengages importin β from the NPC, ensuring receptor turnover and sustaining directional nuclear import.

Ran-GTP influences the permeability of the importin β:α1:NLS-cargo complex as it moves through the NPC. Typically, large cargos require higher levels of Ran-GTP to navigate the NPC, and Ran-GTP primarily regulates the cargo’s exit from the NPC. Without Ran-GTP, cargos can enter and explore the entire NPC, but they are roughly 100 times more likely to exit the NPC on the cytoplasmic side, thus failing to reach the nucleus. NLS-cargos bound to importin α1 require Ran-GTP and the export factor CAS to dissociate on the nucleoplasmic side of the NPC. In contrast, importin α1:NLS-cargo complexes that do not dissociate at the NPC are returned to the cytoplasm. Recent evidence found that import and export complexes share the same central NPC path, rather than distinct lanes. Actively translocating complexes move within a confined ~40–50 nm annulus, while stalled complexes accumulate at the pore periphery.

Ran binding protein 1, or RanBP1, is another key player in the import reaction. It exists as a soluble, 23 kDa protein located in the cytoplasm due to a strong nuclear export signal, although some reports suggest that the protein may shuttle between the cytosol and the nucleus. Four conserved RanBP1-like domains also exist in the filamentous nucleoporin Nup358 at the cytoplasmic periphery of the NPC. A crystal structure revealed that RanBP1 binds Ran-GTP in a tight embrace. The primary function of RanBP1 is to facilitate GTP hydrolysis by RanGAP1, countering the inhibitory activity of transport factors. Without RanBP1, the nanomolar binding affinity of Ran-GTP with importin β inhibits RanGAP1-dependent GTP hydrolysis. However, in the presence of RanBP1, the trimeric importin β:Ran-GTP:RanBP1 complex becomes susceptible to GTP hydrolysis, which additionally requires the importin β binding (IBB) domain of importin α1 in vitro.

Structural and computational studies have been crucial in deciphering the organization, binding interactions, and conformational dynamics of importin β. Several crystal structures have revealed that importin β adopts a solenoid architecture formed by 19 HEAT repeats, organized into two functionally polarized surfaces. Importin β inner (concave) surface, formed by HEAT helix B, binds Ran-GTP and the IBB domain of the adapter importin α, but can also associate directly with nonclassical cargos. Importin β outer (convex) surface, formed by A helices, binds FG-nups. A crystal structure of a fragment of importin β (res 1–442) bound to an FG-rich fragment of nucleoporin Nsp1 reveals two binding sites, one primary site on the A helix of HEAT 5 and another on HEAT 6. This FG-binding pattern was also observed in a structure of Kap95p, the yeast homolog of human importin β, bound to Nup1p. Furthermore, a crystal structure of Kap95p bound to human Ran-GTP revealed an open conformation of Kap95p, in which the C-terminal domain moves away from the closed solenoid seen in the complex with the IBB domain. Based on the conformation of Kap95p observed in this crystal structure, it was proposed that Ran-GTP displaces cargo by inducing a change in the helicoidal pitch of importin β, which opens its concave surface and disassembles the import complex. However, it remains unclear, and is not explained by the above model, how Ran-GTP releases FG-nups from importin β during active transport.

Using cryogenic electron microscopy (cryo-EM) single-particle analysis (SPA), we determine six structures of importin β in a frozen-hydrated state bound to IBB-cargos, FG-nups, Ran-GTP, Ran-GTP:RanBP1, and Ran-GDP:RanBP1. This comparative structural analysis enables us to decipher the conformational plasticity of importin β, overcoming the limitations of previous crystal structures. Our work clarifies how the GTPase Ran-GTP alters the structure of importin β, regulating its binding affinities for cargos and FG-nups.

Results
Molecular dynamics simulations, Förster resonance energy transfer, small-angle X-ray scattering, crystallographic, and biochemical data provided compelling evidence that importin β undergoes large structural fluctuations during nuclear transport. However, most importin β structures have been obtained in the crystalline state in the presence of dehydrating agents that can distort importin β flexible solenoid structure and potentially introduce artifacts. This study aimed to investigate the structure and conformational changes that importin β undergoes during nuclear import under quasi-physiological conditions without potential crystallization-induced artifacts. We formed six complexes of importin β, corresponding to five discrete states observed during the nuclear import reaction. First, we assembled importin β with importin α1 IBB (αIBB) and XRIPαIBB (xIBB), an adapter protein involved in the nuclear import of replication protein A (RPA). Importin βːαIBB and importin βːxIBB are proxies for cytoplasmic importin β assemblies found in both classical and nonclassical import complexes. Second, we added an excess of the FxFG-rich nucleoporin Nsp1 to importin βːαIBB to mimic a classical import complex bound to high-affinity FG sites within the NPC central channel. Third, we assembled the heterodimeric importin β:Ran-GTP complex, which represents the final state of an import reaction presumably formed at the NPC basket, as importin β gets closer to the nucleus. Fourth, we assembled a trimeric importin β:Ran-GTP:RanBP1 complex that was identified nearly three decades ago. And, finally, we reconstituted the heterotrimeric importin β:Ran-GDP:RanBP1 complex, which has also been known for decades but whose function remains unclear.

All importin β complexes were vitrified and imaged using a 300 kV Krios microscope. A total of six cryo-EM reconstructions were determined using SPA, achieving maximal resolutions between 2.6 and 3.4 Å. The corresponding density maps had sufficiently high-resolution features to visualize all side chains, enabling us to build accurate atomic models of importin β bound to different effectors, which were refined to map-to-model correlation coefficients greater than 0.80.

We reconstructed importin β bound to the αIBB domain at 3.25 Å resolution. This structure, determined in a frozen-hydrated state, revealed significant variations compared to the crystal structure of the importin β:αIBB complex (RMSD ~4.7 Å), especially in the C-terminal HEATs 11–19. The N-to-C distance of the X-ray model is 11.5 Å shorter than the cryo-EM structure, suggesting that the constrained, snail-like conformation of importin β:αIBB crystallized at neutral pH represents a nonphysiological state. After establishing that cryo-EM is the preferred methodology for studying importin β curvature, we determined a cryo-EM reconstruction of importin β in complex with xIBB, which we refined to a resolution of 3.41 Å. This structure revealed an elongated solenoid tightly bound to a ~60 Å long peptide, with importin β spanning ~110 Å. The xIBB exhibited strong density for residues 1–45 that contact the concave surface of importin β between HEATs 6–19. A comparison of the two cryo-EM structures of importin β in complex with xIBB and αIBB (solved at comparable resolutions) revealed systematic variations in the solenoid conformation (RMSD ~6.1 Å). The N-to-C distance of importin β bound to xIBB is 9 Å longer than that in complex with αIBB. The two IBBs are highly basic (isoelectric point of ~11) and exhibit a similar topology when bound to importin β, consisting of a long C-terminal α-helix and an unstructured N-terminal moiety. However, xIBB is slightly shorter: its C-terminal α-helix spans six turns of a helix (res. 20–45), compared to seven turns of the αIBB. Both IBBs contain an N-terminal 3/10 helix spanning residues R13–F17 and P8–Y13 in αIBB and xIBB, respectively. The bonding pattern is similar but not identical, primarily driven by electrostatic interactions. xIBB forms five salt bridges, seven hydrogen bonds, and 123 nonbonded contacts with importin β, which is significantly fewer than αIBB, where the binding interface with importin β is stabilized by eight salt bridges, ten hydrogen bonds, and 112 nonbonded contacts. Three tryptophan residues in importin β, W342, W430, and W864, play a crucial role in IBB recognition by making cation-π interactions with IBB arginine and lysine residues. Thus, cryo-EM reconstructions of importin β bound to IBBs reveal that the protein can extend its solenoid structure by up to 10%, repositioning the C-terminal α-helix of different IBBs relative to the HEATs 11–19.

We reconstructed the structure of importin β:αIBB bound to the FG-rich nucleoporin Nsp1 at a maximum resolution of 3.21 Å. The reconstruction revealed strong peaks of density on the convex surface of importin β. We identified five major FG-binding pockets on the convex surface of importin β, formed at the interface between HEAT repeats H4–H6 (pocket 1), H6–H7 (pocket 2), H7–H8 (pocket 3), H9–H10 (pocket 4), and H16–H17 (pocket 5). Pocket 1 binds the PAFSFG motif, positioning the two phenylalanine side chains (F H5 and F H5’) within a bipartite FG-binding pocket between H5 and H6. This pocket is formed by L174, I178, N171, and F217, and represents the previously identified major FG-binding site. A proline residue N-terminal to the FSFG motif (P H4) contacts the interface between H4 and H5. Pocket 2 is also bipartite, and is filled by an AFSF motif, which includes phenylalanine F H6 and F H6’. In contrast, all other pockets are filled with a small density consistent with a single phenylalanine: F H7 for pocket 3, and F H9 and F H16 for pockets 4 and 5, respectively. Like pocket 1, the two smaller pockets 4 and 5 exhibit both hydrophobic and polar residues that interact with the phenyl ring and mainchain atoms. For example, F H16 contacts one hydrophobic residue, L700, one uncharged polar residue, Q741, and one negatively charged glutamate, E703. The side chain oxygen of the glutamate forms a hydrogen bond with the glycine main chains and has a nonbonded contact with the F H16 side chain. Thus, the chemistry of FG-binding to importin β includes both hydrophobic and polar contacts, suggesting FG-binding pockets are hydrated when importin β is not associated with FG-nups.

The cryo-EM reconstruction presented here identified all major FG-binding major sites (F H5 and F H6) described crystallographically and probed biochemically (F H16). Additionally, we experimentally validated five of the eight FG sites identified in silico using molecular dynamics simulations, all of which are located on the convex surface of importin β. Importantly, we found that the conformation of importin β observed in our cryo-EM reconstruction bound to FGs is similar to the importin:αIBB complex, with an RMSD of approximately 1.9 Å. This indicates that FG-binding does not significantly alter the protein solenoid, at least in the αIBB-bound conformation.

We elucidated the cryo-EM structure of the importin β:Ran-GTP complex at a resolution of 3.34 Å. In this structure, importin β is significantly closed around Ran-GTP, making extensive contact with HEATs 1–13. Specifically, Ran-GTP engages at three contact points with the concave surface of importin β, at HEATs 1–4 (contact point a), HEATs 6–8 (contact point b), and HEAT 13 (contact point c). Ala178 is the last residue of Ran-GTP with discernable density in our reconstruction, whereas C-terminal residues 179–216 are disordered. The association between importin β and Ran-GTP comprises 10 salt bridges and 11 hydrogen bonds, burying 2388 Å2 of the importin β concave surface. Perhaps the most striking finding is that the cryo-EM structure of the human importin β:Ran-GTP complex differs dramatically from the crystal structure of the Kap95p:Ran-GTP complex (PDB: 2BKU) with a staggering RMSD of 7.4 Å. In the cryo-EM structure, the C-terminus of importin β is completely closed relative to Ran-GTP, which is precisely the opposite of what is seen in the crystal structure of Kap95p:Ran-GTP. The main difference between cryo-EM and crystal structures is that Kap95p:Ran-GTP crystallizes as a tight dimer stabilized by intermolecular dimerization of HEAT repeats 15–19, which forces the solenoid into an elongated conformation. In contrast, in vitreous ice, the importin β solenoid bound to Ran-GTP is under conformational strain, with N- and C-termini showing a maximum displacement of 19.4 Å compared to 40.4 Å in the Kap95p:Ran-GTP complex. Superimposing the cryo-EM structure of importin β with the Kap95p X-ray model reveals that the latter has open, relaxed, and untwisted C-terminal HEATs 17–19 with a displacement of approximately 25 Å relative to the equivalent HEAT repeats in the human homolog. Thus, the previous crystal structure of Kap95p bound to Ran-GTP adopts a tertiary structure that is drastically different from that of human importin β.

Importin β and RanBP1 do not bind to each other without Ran, but the three proteins form a complex together, which accelerates Ran-GTP hydrolysis in the presence of importin α. We assembled importin β with RanQ69L, which is trapped in a GTP-bound state, along with RanBP1, and reconstructed this trimeric complex using cryo-EM SPA at 2.62 Å resolution. Unexpectedly, we found RanBP1 inserts itself at the interface between importin β and Ran-GTP, predominantly binding to the outer surface of Ran-GTP. The GTPase C-terminal moiety (res. 191–216) wraps around the RanBP1 core, recapitulating the tight embrace observed in the Ran-GTP:RanBP1 crystal structure. Notably, RanBP1 makes limited contact with importin β in the ternary complex, limited to just one nonbonded interaction between R44 and H520. Nonetheless, the association of RanBP1 with Ran-GTP profoundly influences the importin β solenoid, causing it to relax and extend, adopting a conformation that is even more elongated than when bound to xIBB. RanBP1 alters the association of importin β with Ran-GTP in three significant ways. First, the C-terminal helix of Ran-GTP, disordered in the importin β:Ran-GTP complex, becomes structured with RanBP1, disrupting a salt bridge between Ran-GTP R15 and importin β E46 in HEAT 1. Second, the binding interface between RanBP1 and Ran-GTP weakens the importin β:Ran-GTP binding interface at contact point c, resulting in the loss of two salt bridges and three hydrogen bonds. This is offset by 10 nonbonded interactions between RanBP1 R44 and HEAT 12 H520. Third, the importin β:Ran-GTP association at HEAT 13 is partially disrupted due to a dramatic structural rearrangement of HEAT 13. To accommodate both RanBP1 and Ran-GTP, HEAT 13 residues 542–595 bend by 90 degrees in the ternary complex, forming intramolecular contacts with the concave surface of importin β.

Thus, RanBP1’s association with importin β:Ran-GTP weakens two of the three bonding regions between the two proteins, specifically, points a and c, while keeping the bonding contacts between HEATs 2 and 8 seemingly unchanged. In the presence of RanBP1, the interaction between Ran-GTP and importin β decreases to six salt bridges and seven hydrogen bonds, with a total burial of 2038 Å2 of importin β‘s concave surface. Notably, the entire C-terminal cluster, consisting of HEATs 14–19, swings away from Ran-GTP in the presence of RanBP1, relaxing the solenoid in a way that contrasts significantly with the closed, snail-like conformation observed in the complex with Ran-GTP alone.

Both in vitro and in live cells, importin β forms a stable complex with Ran-GDP and RanBP1. The function of this complex is poorly understood, and the biochemical determinants responsible for its association are unclear, given that importin β binds Ran-GDP weakly (Kd ~2 μM), and RanBP1 exhibits undetectable binding to Ran-GDP but interacts strongly with Ran-GTP. To decipher how importin β can associate with both Ran-GDP and RanBP1, we assembled a stoichiometric complex of the three proteins, which we subjected to cryo-EM SPA and used to reconstruct at 3.21 Å resolution. Notably, the reconstruction of the trimeric complex shows density only for importin β residues 1–459, which include HEAT repeats 1–10, along with the full Ran-GDP:RanBP1 heterodimer. In the ternary complex, importin β has a flexible C-terminus, with HEATs 11–19 not visible in the reconstruction due to motion and lack of binding to Ran-GDP:RanBP1. Ran-GDP adopts a Ran-GTP-like conformation that makes nearly identical contacts with Ran-BP1 as seen in the importin β:Ran-GTP:RanBP1 complex. A similar GTP-like conformation of Ran-GDP was observed in a crystal structure of the yeast Kap95p:Ran-GDP complex, suggesting that importin β can stabilize Ran’s conformation regardless of its nucleotide-bound state. In complex with RanBP1, Ran-GDP’s C-terminal moiety (res. 191–216) wraps around the RanBP1 core, embracing RanBP1. However, the Ran-GDP:RanBP1 heterodimer has only two contact points with importin β, at HEATs 1–4 and 8–9, but does not associate with HEAT 13. Because of this loss of binding, the C-terminus of importin β does not coil around the Ran-GDP:RanBP1 complex as it does with Ran-GTP, but instead remains flexible, likely resembling unliganded importin β, which has proven intractable for cryo-EM single-particle analysis.

To rationalize the unexpected open structure of importin β in complex with Ran-GDP:RanBP1, we superimposed the Ran-GDP:RanBP1 and Ran-GTP:RanBP1 heterodimers bound to importin β. The global RMSD is only ~1.9 Å, suggesting that the overall conformation is highly similar. Subtle changes exist in switch loop I (res. 32–43) and switch loop II (res. 66–84) of Ran that surround the nucleotide. In Ran-GDP:RanBP1, the basic patch region (res. 122–138) is shifted outward by 5 Å relative to the GTP-bound state, toward the position occupied by HEAT 13 in the importin-β Ran-GDP:RanBP1 complex. This expansion of the Ran-GDP basic patch is driven by a series of small shifts triggered by the absence of the γ-phosphate in GDP, which causes the nucleotide to move downward between the switch loops, thereby pushing the basic patch outward.

Overall, DynDom analysis of importin β domain motion in response to binding effectors identified three clusters of HEAT repeats. Two moving clusters comprising N-terminal HEATs 1–4 (cluster I) and C-terminal HEATs 11–19 (cluster III), along with a fixed cluster II formed by HEATs 5–10, which is invariant in all our cryo-EM structures. Clusters I and III move relative to cluster II around the helices B of HEAT 5 (bending helix I) and HEAT 10 (bending helix III). Ran-GTP binds to importin β, causing a 16-degree clockwise rotation in moving cluster I (H1–H4) and a 10-degree rotation in moving cluster III (H11–H19) while maintaining a fixed configuration in the region H5–H10. These rotations are linked to bending at the N- and C-termini around the helix B of H5 and H10. Because the N- and C-terminal clusters also twist around the two helices, the domain movement of clusters I and III is better described as bending rather than a hinge movement. Notably, importin β cluster III is fully mobile when complexed with Ran-GDP:RanBP1, where the rotation of cluster III relative to cluster II may resemble that of unbound importin β. The two movements of H1–H4 and H11–H19 must account for the known activities of Ran-GTP, namely, reducing FG-nup binding avidity and displacing cargo from importin β.

Ran-GTP reduces the affinity of importin β for FG-nups; however, none of the crystal structures determined to date have explained how this occurs. To address this question, we focused on the outer surface of frozen-hydrated importin β, which was solved in complex with αIBB:FG-repeats, Ran-GTP, Ran-GTP:RanBP1, and Ran-GDP:RanBP1. Guided by the atomic coordinates of FGs bound to importin β:αIBB, we measured the volume and solvent-accessible surface area (SASA) of each of the five pockets in importin β using FPocketWeb. We found that the five pockets have volumes ranging from 502 to 154 Å3, displaying characteristics that lie between pocket-like invaginations and cavities. We then compared the volume and shape of these FG-binding pockets in importin β bound to Ran-GTP, Ran-GTP:RanBP1, and Ran-GDP:RanBP1. Strikingly, we observed a substantial size reduction in cavity volume and SASA in two pockets: pocket 1 (H4–H6) and pocket 4 (H9–H10). Notably, the bipartite pocket between H5–H6 displayed a significant decrease in solvent-accessible volume at the first site (H5–H6), diminishing from 502 to 122.0, 303, and 291 Å3, respectively. This site is likely vital, as a single point mutation at I178D abolishes importin β nuclear import in permeabilized cells. Similarly, the pocket between H9–H10 (pocket 4) that binds F H9 exhibited a noteworthy reduction in solvent-accessible volume, with the F H9 pocket decreasing from 500 to 194, 325 and 346 Å3. Notably, F H9 binds at the bending region identified when Ran-GTP or Ran-GDP, are associated with importin β. Additionally, pocket 5 located between H16–H17 at the C-terminal end undergoes a remarkable decrease in solvent-accessible volume only in the importin β:Ran-GTP:RanBP1 complex, diminishing from 545 to 290 Å3. This pocket is not visible in the complex with Ran-GDP:RanBP1 that has a disordered C-terminus. Finally, Ran-GTP, Ran-GTP:RanBP1, and Ran-GDP:RanBP1 binding to importin β did not perturb pockets 2 and 3, which are very shallow.

We also examined the FG-binding pockets of Kap95p identified in the crystal structure with Nup1 and compared them to those in the crystal structure with Ran-GTP to detect changes in volume and SASA. Notably, Nup1 contacts three FG-pockets in Kap95p outer surface, which are analogous to pockets #1, #2, and #4 in importin β. The volume of these pockets was surprisingly reduced in complex with Ran-GTP. Especially, the pocket #1 (H5–H6) and #3 (H7–H8) display a significant decrease in solvent-accessible volume, which drops from 459 to 228.0 Å3 and 463 to 221 Å3, respectively, when Ran-GTP is bound. The pocket #2 instead did not have significant changes in solvent-accessible volume and SASA. Therefore, in solution as well as in the crystalline state, Ran-GTP binding to the inner surface of importin β and Kap95p induces an allosteric change on the outer surface that closes the primary FG-binding pockets, located between HEAT repeats H4–H6, H9–H10, and H16–H17 in importin β, and between H5–H6 and H7–H8 in Kap95p.

We experimentally validated the prediction that importin β bound to RanGTP:RanBP1 exhibits reduced binding affinity for FG-nucleoporins, as expected for the RanGTP-bound state. To this end, we immobilized GST-tagged Nup358, Nup62, and Nup153 on glutathione beads, representing FG-nups localized to the cytoplasmic face, central channel, and nuclear basket of the NPC, respectively. In an initial set of pull down assays, all three FG-nups efficiently captured free importin β as well as importin β in complex with αIBB. Notably, Nup62 bound free importin β slightly more strongly than the importin β:αIBB complex, consistent with a previous report showing that importin β affinity for Nup62 decreases by up to 40-fold when bound to αIBB. By contrast, no statistically significant difference was observed between importin β alone and αIBB–IBB to Nup358 or Nup153. In the presence of Ran-GTP alone or Ran-GTP plus RanBP1, importin β exhibited dramatically reduced binding to all tested FG-nups. Ran-GTP and Ran-GTP:RanBP1 dissociated ~90–95% of all importin β from GST-Nup62 and -Nup358, whereas about 20–25% of importin β remained bound to GST-Nup153. No statistically significant difference was observed between Ran-GTP alone and Ran-GTP-RanBP1, both of which equally displaced importin β from FG-nups. Therefore, Ran-GTP binding to the concave surface of importin β induces a long-distance conformational change, which reduces the solvent-accessible surface of at least two FG-binding pockets on the outer surface, thereby disrupting FG association in vitro. The ternary importin β:Ran-GTP:RanBP1 complex exhibits a low affinity for FG-nups due to the closure of three FG-binding pockets 1, 4, and 5.

We then repeated the pull-down assay described above using Ran-GDP, both alone and together with RanBP1. As expected, Ran-GDP alone did not significantly alter the association of importin β with FG-nups. Notably, the addition of RanBP1 with Ran-GDP did not affect the binding of importin β to Nup358. Still, it produced a very small yet statistically significant reduction in binding to Nup62 and Nup153. This modest decrease in affinity may be explained by the partial closure of the FG-binding pockets #1 and #4 observed in the structural model displayed in Fig. 8B.

We next investigated the IBB interface for IBB and Ran-GTP and identified three binding regions: HEATs 1–6 (region I), HEATs 7–13 (region II), and HEATs 14–19 (region III). These three regions are similar but not identical to the three moving clusters identified through local domain motion analysis. Both αIBB and xIBB domains do not contact region I, form only two salt bridges with region II, and create over ten hydrogen bonds with importin β C-terminal HEATs 14–19, which is part of region III. In contrast, Ran-GTP primarily binds to importin β through the N-terminal regions I and II. The interaction with importin β involves five hydrogen bonds in region I and five salt bridges along with six hydrogen bonds in region II. Residues E281 and D288 in region II are the only residues in importin β bound by either IBB or Ran-GTP, both engaging in electrostatic interactions. Thus, the binding sites for IBBs and Ran-GTP in importin β are mainly distinct. IBB associates primarily with the C-terminal region III (HEATs 7–19), whereas Ran-GTP binds to the N-terminal arch comprising HEATs 1–13, validating previous mutagenesis studies, with some overlap in HEAT 13. Strikingly, in the presence of Ran-GTP:RanBP1, three salt bridges and three hydrogen bonds between Ran-GTP and importin β in region II are disrupted by RanBP1, resulting in an overall opening of HEATs 14–19, potentially creating space to accommodate the IBB.

We then asked whether RanBP1 would facilitate the formation of a larger assembly comprising both αIBB and Ran-GTP. We titrated an equimolar quantity of purified Ran-GTP against 5 μg of importin β and visualized the relative association using native PAGE. We assembled the importin β:Ran-GTP:RanBP1 complex by adding a stoichiometric amount of purified RanBP1, resulting in a ternary complex that shifted upward compared to importin β:Ran-GTP. Finally, we titrated a 0–10-fold excess of αIBB fused to the maltose binding protein (MBP-αIBB) against the importin β:Ran-GTP:RanBP1 ternary complex. Although the MBP-αIBB chimera is basic and does not migrate well on a native gel, it significantly shifted the migration of the trimeric importin β:Ran-GTP:RanBP1 complex. In the presence of MBP-αIBB, a new band shifted upwards was observed, corresponding to a tetrameric complex formed by importin β:Ran-GTP:RanBP1:MBP-αIBB. Quantification of native gel bands indicated that about half (e.g., 52.1 ± 12.8%) of the trimeric importin β:Ran-GTP:RanBP1 complex was converted into a tetrameric complex upon addition of a 4-fold molar excess of αIBB. This tetrameric importin β:Ran-GTP:RanBP1:αIBB complex was not observed when the αIBB was titrated against a preformed importin β:Ran-GTP without RanBP1. Therefore, the binding of RanBP1 to importin β and Ran-GTP stabilizes a conformation of the importin β C-terminus that is capable of binding αIBB, which in this study serves as a proxy for the classical import complex (e.g., importin α1:NLS-cargo).

We also repeated the titration in Fig. 10B using Ran-GDP, which, as expected, bound importin β and RanBP1, forming a trimeric importin β:Ran-GDP:RanBP1 complex. However, adding up to a 10-fold molar excess of MBP-αIBB to a preformed importin β:Ran-GDP:RanBP1 complex did not cause a further upward shift on the native gel, indicating that the importin β:Ran-GDP:RanBP1 complex cannot bind cargo. Overall, this data suggests that the importin β:Ran-GDP:RanBP1 complex is not directly involved in mediating the nuclear import of NLS cargos through the NPC.

Methods
The genes encoding the importin β:α1 heterodimer were cloned into the vector pACYCDuet-1 (EMD Millipore) with importin α1 fused to an N-terminal 6×His-tag and importin β as untagged. The proteins were expressed in BL21-DE3 Escherichia coli (E. coli) cells. Protein expression was induced with 500 µM IPTG for 3 h at 30 °C, and the heterodimer was purified to homogeneity using metal affinity chromatography and size-exclusion chromatography (SEC) using a Superdex 200 16/600 preparative column (Cytiva). Untagged importin β was separated from the importin β:α1 complex bound to the nickel agarose beads (GenScript) using a Separation Buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 250 mM MgCl2, and 3 mM 2-mercaptoethanol). Importin β was further purified by SEC on a Superdex 200 26/600 preparative column (Cytiva) equilibrated in β SEC Buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM PMSF, and 3 mM 2-mercaptoethanol).

The importin α1 IBB domain (res 1–65) C-terminally fused to a 6xHis Maltose Binding Protein (MBP-αIBB) was cloned by PCR into an engineered pET28a vector that also contains the MBP gene downstream of the 6xHis tag, using the following forward (5′-CAGCCGGATCCATGTCCACCAACGAGAATGCTA-3′) and reverse (5′-CAGCCAAGCTTCTACTGCAGCGGAGAAGTAGCATC-3′) oligos. MBP-αIBB was expressed in BL21-DE3 E. coli cells. Protein expression was induced with 500 µM IPTG for 3 h at 30 °C. Cells were resuspended in IBB Lysis Buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM PMSF, 3 mM 2-mercaptoethanol, 1 mM EDTA, and 0.1% Triton X-100, DNase, and RNase) and after sonication, the soluble fraction was bound to amylose resin (New England Biolabs). The protein was washed using a lysis buffer and then eluted with a lysis buffer with 15 mM maltose. MBP-αIBB was further purified by SEC using a Superdex 200 16/600 preparative column (Cytiva) equilibrated with IBB SEC Buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl).

Ran-GTP was expressed and purified using a Ran-Q69L mutant that remains in a GTP-bound conformation. Ran-Q69L was cloned into a pET28a-PPase construct with an N-terminal 6xHis tag and expressed in BL21-DE3 E. coli strain supplemented with kanamycin. Protein was expressed overnight at 18 °C with 500 µM IPTG. Cells were resuspended in Ran Lysis Buffer (50 mM KPO4 pH 7.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, and 3 mM 2-mercaptoethanol) and, after sonication, the soluble fraction was bound to low-density nickel agarose beads (GoldBio). The protein was washed using a lysis buffer containing 10 mM imidazole and then eluted with a lysis buffer with Ran Elution Buffer (50 mM KPO4, pH 7.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, 3 mM 2-mercaptoethanol, and 150 mM imidazole). Ran-GTP was further purified by SEC using a Superdex 200 16/600 preparative column (Cytiva) equilibrated with Ran SEC Buffer (50 mM KPO4 pH 7.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, and 3 mM 2-mercaptoethanol). The importin β:Ran-GTP complex was formed by incubating importin β and Ran-GTP at 1–4 molar ratio at 4 °C for 30 min.

To produce Ran-GDP, we expressed the full-length human Ran cloned into a pET28a-PPase vector with an N-terminal 6xHis tag in BL21-DE3 E. coli. Protein was expressed overnight at 18 °C with 500 µM IPTG. Cells were resuspended in Ran-GDP Lysis Buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, and 3 mM 2-mercaptoethanol) and, after sonication, the soluble fraction was bound to low-density nickel agarose beads (GoldBio). The protein was washed using a lysis buffer containing 10 mM imidazole and then eluted with a lysis buffer with Ran-GDP Elution Buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, 3 mM 2-mercaptoethanol, 150 mM imidazole). The Ran protein was incubated with 2 mM GDP on ice for 30 min before SEC. Ran-GDP was further purified by SEC using a Superdex 200 16/600 preparative column (Cytiva) equilibrated with Ran-GDP SEC Buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 2 mM MgCl2, 1 mM PMSF, and 3 mM 2-mercaptoethanol). The importin β:Ran-GDP:RanBP1 complex was formed by pre-incubating importin β and Ran-GDP and RanBP1 at 4 °C for 30 min and purified by SEC using a Superose 6 increase 10/300 GL column (Cytiva) equilibrated with Ran-GDP SEC Buffer.

RanBP1 was cloned into a pET28a-PPase vector with an N-terminal 6xHis tag and expressed in BL21-DE3 E. coli cells. Protein expression was induced with 500 µM IPTG for 3 h at 30 °C. Cells were resuspended in a Low Salt Lysis Buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM PMSF, and 3 mM 2-mercaptoethanol), lysed by sonication, and the soluble fraction isolated by centrifugation and incubated with low-density nickel agarose beads (GoldBio). The protein was washed using a low salt lysis buffer containing 5–20 mM imidazole, 50 mL for each wash, then eluted with Elution Buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM PMSF, and 3 mM 2-mercaptoethanol, 150 mM imidazole). RanBP1 was further purified by SEC using a Superdex 200 16/600 preparative column (Cytiva) equilibrated with PBS plus 0.1 mM PMSF.

The FxFG-rich fragment (residues 497–608) of yeast Nsp1 was cloned into expression vector pMW172 with an N-terminal 6xHis tag. The protein was expressed for 3 h at 30 °C after adding 400 µM IPTG to cells with an optical density at 600 nm of ~0.6. The Nsp1 fragment was purified in a High Salt Lysis Buffer (20 mM Tris-HCl, pH 8.0, 600 mM NaCl, 1 mM PMSF, and 3 mM 2-mercaptoethanol and 5% glycerol) and bound to low-density nickel agarose beads (GoldBio). The protein was washed with a high salt buffer containing 5/10/20 mM imidazole, 10 mL for each wash, then eluted with Elution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM PMSF, and 3 mM 2-mercaptoethanol, 150 mM imidazole) supplemented with 1.25% glycerol. The importin β:α1:FG complex was formed by adding a 2-fold molar excess of Nsp1 (497–608) to purified importin α1/β and used immediately for vitrification.

GST-tagged constructs of Nup358 (res. 2503–2893), full-length Nup62, and Nup153 (res. 946–1472) were cloned into the vector pGEX-4T-1 (Cytiva) and expressed in BL21-DE3 E. coli cells. Protein expression was induced with 500 µM IPTG for 3 h at 30 °C. Cells were resuspended in a Medium Salt Lysis Buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.2% Triton X-100, 1 mM PMSF, and 3 mM 2-mercaptoethanol) plus protease inhibitor, and after sonication, the soluble fraction was bound to glutathione beads (GenScript). The protein was washed using a lysis buffer containing 0.2% Triton X and then buffer-exchanged to Protein Binding Buffer (20 mM HEPES pH 7.0, 150 mM KOAc, 2 mM Mg(OAc)2, 1 mM dithiothreitol, and 0.1% Tween) plus protease inhibitors. The immobilized protein was used immediately to avoid degradation. A peptide comprising XRIP1α residues 6–47 (xIBB) was synthesized by GenScript.

GST-tagged fragments of Nup358 (res. 2503–2893) and Nup153 (res. 946–1472), as well as full-length p62 (res. 1–522) were immobilized on glutathione beads (GoldBio) and resuspended in Pulldown Binding Buffer (20 mM HEPES pH 7.0, 150 mM KOAc, 2 mm Mg(OAc)2, 1 mM dithiothreitol, and 0.1% Tween 20). Pulldown assays were carried out as described. Briefly, batches of 50 μL of glutathione beads coupled with GST-Nups were incubated with either 3 μM importin β alone, or (3 μM importin β + 3 μM MBP-αIBB), or (3 μM importin β + 3 μM Ran-GTP), or (3 μM importin β + 3 μM Ran-GTP + 3 μM RanBP1). All reactions were incubated for 1 h at 4 °C. Unbound fractions were washed away, and beads were washed three times with 150 μL of Pulldown Wash Buffer (20 mM HEPES pH 7.0, 150 mM KOAc, 2 mM Mg(OAc)2, 1 mm dithiothreitol, and 0.1% Tween 20). The entire pool of 50 μL of washed beads that contains the bound fractions was resuspended in 25 μL of 4× SDS-sample buffer and 5 μL was analyzed on an 8–16% Tris-Glycine mini protein gel (Invitrogen, XP08162BOX). SDS-PAGE gels were run across four independent experiments (n = 4), and the average ratio of importin β band intensity normalized to each GST-FG-nup was determined through densitometry using ImageJ. We employed a two-tailed Student’s t test, assuming unequal variances, to determine statistical significance between the means of the control and experimental pulldowns. The alpha parameter had to be outside the 99th percentile for statistical significance (p < 0.01). Error bars indicate the standard deviation of the mean.

Native Gel electrophoresis was performed using NativePAGETM Bis-Tris Mini Protein Gels, 3–12% (Invitrogen, BN1001). Briefly, 5 μg of importin β or importin β complexes were incubated on ice for one hour, with increasing molar excesses (typically between 0.5 and 10×) of Ran-GTP (or Ran-GDP), RanBP1, and/or αIBB. After 1 h, 3.75 μL of 4× Loading buffer (Invitrogen, BN2003) was added to the sample to reach a final volume of 15 μL. Samples were then loaded onto a 3–12% Bis-Tris native gel, run the gel with Bis-Tris non-denaturing running buffer (Invitrogen, BN2007) at 150 V for 90 min, at 4 °C. The gel was stained with Coomassie Brilliant Blue-G-250 and destained overnight in 50% (v/v) methanol in water with 10% (v/v) acetic acid.

For all importin β complexes studied in this paper, typically 2.0 µl of importin β pre-bound to αIBB, xIBB, αIBB:Nsp1, Ran-GTP, Ran-GTP:RanBP1, or Ran-GDP:RanBP1 at 2 mg/ml final concentration were applied to a 300-mesh copper Quantifoil R1.2/1.3 holey carbon grid (EMS) previously glow-discharged negatively for 60 s at 15 mA using an easiGlow (PELCO). Only for the importin β:αIBB:Nsp1 complex (referred to here as importin β:αIBB:FG) and importin β:Ran-GTP:RanBP1, we used 200-mesh UltraAUfoil R1.2/1.3 grid (EMS) instead of standard carbon grids. All grids were blotted for 5 s at blot force 5 and frozen in liquid ethane using Vitrobot Mark IV (FEI). All micrographs were pre-screened in-house on a 200 kV Glacios2 equipped with a Falcon 4i detector at the UAB Cryo-EM Facility. EPU (v3.10) software was used for data screening and collection using the accurate positioning mode. All high-resolution datasets were collected at Titan Krios microscopes available at three federal facilities equipped with either a K3 direct electron detector camera (e.g., NCEF and NCCAT) or a Falcon 4i (e.g., S2C2 and NCCAT). The software used for data collection was SerialEM (v4.2.10) for Gatan K3 and EPU (v3.10) for Falcon 4i. 9497 micrographs were collected for importin β:xIBB in super-resolution mode plus energy filter at 20 eV with an image pixel size of 0.436 Å at ×105,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and defocus range −1.0 to −2.5 μm. 27,987 micrographs were collected for importin β:αIBB in super-resolution mode plus energy filter at 20 eV with an image pixel size of 0.371 Å at ×165,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and defocus range −0.8 to −2.5 mm. 18,016 micrographs were collected for importin β:αIBB:FG plus energy filter at 20 eV with an image pixel size of 0.959 Å at ×130,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and defocus range −0.8 to −2.5 μm. 13,468 micrographs were collected for importin β:Ran-GTP in super-resolution mode with an energy filter at 20 eV, using an image pixel size of 0.436 Å at ×105,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and a defocus range of −1.0 to −2.5 μm. 11,100 micrographs were collected for importin β:Ran-GTP:RanBP1 plus energy filter at 20 eV with an image pixel size of 0.371 Å at ×165,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and defocus range −0.8 to −2.5 μm. 22,555 micrographs were collected for importin β:Ran-GDP:RanBP1 plus energy filter at 20 eV with an image pixel size of 0.371 Å at ×165,000 magnification, a nominal total dose of 50 e/Å2, 40 frames, and defocus range −0.6 to −3.7 μm. All collection parameters are in Supplementary Table 1.

Micrographs were subjected to patched motion-correction using MotionCor2 (v1.6.4) and patched contrast transfer function (CTF) estimation using CTFFIND4 (v4). All subsequent steps of SPA were carried out using cryoSPARC version 4.2.1. Particle picking was done using one round of template picker and at least three rounds of Topaz training and extraction, followed by extracting particles from micrographs. In total, we picked 4.3 million for importin β:xIBB, 11.9 million for importin β:αIBB, 5.8 million particles for importin β:αIBB:FG, 6.5 million for importin β:Ran-GTP, 9.6 million for importin β:Ran-GTP:RanBP1, and 1.81 million for importin β:Ran-GDP:RanBP1. The initial maps were reconstructed by using a combination of selected 2D classes and ab initio reconstruction. In total, 293,677, 157,234, 593,354, 1,400,279, 721,883, and 241,237 particles were used to calculate 3D densities for importin β:xIBB, importin β:αIBB, importin β:αIBB:FG, importin β:Ran-GTP, importin β:Ran-GTP:RanBP1, and importin β:Ran-GDP:RanBP1, respectively. The 3D maps were further refined using heterogeneous 3D classification, homogeneous refinement with CTF and defocus refinement options, and post-processing on a 10-GPU cluster. The final densities were sharpened using phenix.auto_sharpen. A flowchart of cryo-EM SPA methods used in this paper is displayed in Supplementary Fig. 1.

All atomic models were built using Coot (v0.9.8.7) and ChimeraX (v1.9) followed by several rounds of rigid-body, real-space, and B-factor refinement using phenix.real_space_refinement in Phenix (v1.21). All final models were validated using MolProbity (Supplementary Table 1). All images of models and cryo-EM maps were generated using ChimeraX (v1.9) and PyMol (v2.3). RMSD between superimposed PDBs, structural comparison, and domain motion analysis were done using the DynDom server and SuperPose 1.0. FPocketWeb was used to identify and characterize binding pockets within importin β. Binding interfaces were analyzed using PISA and PDBsum.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Ko et al. investigate how the GTPase Ran regulates the nuclear import process mediated by importin-β. Utilizing cryo-electron microscopy (cryo-EM), the authors resolve four distinct conformational states of importin-β in complex with various effectors, including IBB-cargos, FG-repeats, Ran-GTP, and Ran-GTP:RanBP1. By comparing these structures and integrating biochemical data, they demonstrate that Ran-GTP induces an allosteric constriction of importin-β’s solenoid structure, closing high-affinity FG-binding pockets and promoting cargo release. This structural biology-oriented manuscript elucidates an allosteric mechanism by which Ran-GTP modulates importin-β function, with broader implications for other β-karyopherins involved in nuclear transport.\nPrevious structures of importin-β in complex with IBB domains, FG-repeats, and Ran-GTP were determined by X-ray crystallography under dehydrating conditions. Given the inherent flexibility of solenoidal structures, crystallization can potentially induce distortions and artifacts. Therefore, the authors leverage cryo-EM, which better preserves near-physiological conditions, to investigate the conformational changes of importin-β during cargo binding and to propose an updated model for importin-β-mediated nuclear import.\nA particularly interesting observation from the cryo-EM structures is that FG-binding pockets on importin-β undergo a significant decrease in solvent-accessible volume upon binding Ran-GTP or Ran-GTP:RanBP1, limiting the receptor's re-entry into the nuclear pore. However, several critical issues must be addressed to fully substantiate the physiological relevance of the proposed model.\nMajor concerns:\nThe cryo-EM structures of the IBB–importin-β complexes appear substantially more elongated compared to prior X-ray structures. Given the known flexibility of importin-β, the observed conformations may represent only a subset of possible configurations. Furthermore, the FG-repeat-bound importin-β structure appears structurally similar to the IBB-bound form. The authors report selecting ~4.3 million to ~11.9 million particles for initial processing across different complexes, but the final reconstructions are based on significantly fewer particles (e.g., 293,677 for importin-β–xIBB and 1,400,279 for importin-β–Ran-GTP). This discrepancy raises concerns about particle heterogeneity and whether the resolved structures represent physiologically dominant states. It would be informative to assess whether the selected particles reflect a major or minor population and to discuss the implications for physiological relevance. In addition, comparison of FG-repeat-bound importin-β structures obtained by both cryo-EM and X-ray crystallography would strengthen the argument.\nThe cryo-EM structure of the RanGTP–importin-β complex reveals a closed configuration, particularly at the C-terminus, which differs from the open conformation observed in the Kap95p–RanGTP complex. It is critical to determine whether crystal structures of RanGTP–importin-β also show reduced solvent accessibility of FG-binding pockets. Furthermore, since the decrease in solvent-accessible volume upon RanGTP binding is a key finding, functional validation through mutagenesis, followed by cell-based assays, is necessary to test the physiological impact of this structural change.\nA major concern is the stability and physiological relevance of the RanBP1–importin-β–RanGTP complex. RanBP1 is known to facilitate the dissociation of RanGTP from importin-β, promoting complex disassembly and recycling of importin-β. Therefore, the trimeric complex is expected to be transient and highly dynamic, not forming a stable assembly under physiological conditions. The complex observed in vitro likely reflects an artificial state induced by experimental conditions. The authors should discuss this discrepancy and explicitly address the likely non-physiological nature of the RanBP1–importin-β–RanGTP complex.\nIn Figure 8, it is shown that importin-β alone binds FG-Nups more strongly than cargo-bound importin-β. The authors should clarify whether importin-β alone can shuttle through the nuclear pore independently. Moreover, given that unliganded importin-β cannot efficiently interact with RanBP1, how is its re-entry into the nuclear pore regulated?\nMinor issues:\n1. Figure 8 (SDS-PAGE): The SDS-PAGE gel in Figure 8 is inadequately labeled (e.g., molecular weight markers).\n1. Importin-β Protein Disappearance in Pull-Down Assays (Figure 8):\nIn the pull-down assays using GST-tagged FG-Nups, importin-β protein seems to disappear in both the bound and unbound fractions (lanes 4–6 and 10–12). Moreover, a quantitative analysis plotting the ratio of importin-β in bound fractions over total input should be provided to ensure consistent protein loading across samples.\n2. GST-tagged Nup358 (residues 2503–2893), full-length Nup62, and Nup153 (residues 946–1472) should have distinct molecular weights. However, they appear at comparable positions on the SDS-PAGE gel (Figure 8).\n3. Figure 9 (Native Gel and Quantification): In Figure 9, if IBB binds to the trimeric complex (importin-β–RanGTP–RanBP1), the intensity of the trimeric complex should decrease. However, this reduction is not evident. The authors should provide a quantitative densitometry analysis to support their interpretation.\n4. Interpretation of Figure 9B: The conclusion that \"association of RanBP1 with importin-β and RanGTP promotes binding to αIBB\" is unclear, especially since RanGTP typically promotes dissociation of importin-α/β–cargo complexes. A more detailed mechanistic explanation is required to reconcile these findings.\n5. Validation of FG-Nup Displacement by αIBB Binding (Competition Assay):\nIn the Discussion, the authors propose that a transient importin-β:α1:NLS-cargo complex, when simultaneously bound to RanGTP and RanBP1, exhibits reduced avidity for FG-Nups, facilitating passage through the NPC. However, the current experiments primarily demonstrate that αIBB can bind to the RanGTP–RanBP1–importin-β complex. To directly test the physiological relevance of the model, it is important to perform a competition assay to determine whether αIBB binding actively displaces pre-bound FG-Nups from importin-β. Such an experiment would provide more definitive evidence supporting the authors’ proposed mechanism.", "reviewer_2": "Ko and colleagues address a long standing gap in the understanding of mobile nucleocytoplasmic transport factors, using cryo-electron microscopy (cryoEM) to resolve the structural changes observed between a comprehensive set of binding configurations for the prototypical import karyopherin human Importin-beta (or Kapbeta1). The authors present a well-balanced structural and biochemical dissection of Importin-beta, determining five high quality cryoEM structures (between 2.6-3.4 Å resolution): bound to either IBB domains of the cargo adapters Importin-alpha or xRIP-alpha, FG repeat peptides that mimic the diffusion barrier during transit across the nuclear pore complex (NPC), or in complex with either Ran(GTP) alone or a Ran(GTP)•RanBP1 complex, highlighting nuclear cargo release and cytoplasmic Ran hydrolysis states, respectively. Within this impressive set of structures are the first full-length structures of importin-beta in complex with FG-peptides, demonstrating additional binding sites that bring the structurally resolved FG epitopes from two to seven, and crucially validate the existence of several minor sites which where only previously indicated by biochemical mutation and in silico approaches. Additionally, the authors solve a structure of the Importin-beta•Ran(GTP)•RanBP1 pre Ran hydrolysis recycling intermediate, with native-PAGE gels indicating that this configuration can combine both Ran(GTP) and cargo binding simultaneously, tying cargo release to defined locations proximal to the NPC where there is an abundance of available Ran binding domains including RanBP1. However, the stand out observation is that unlike earlier crystal structures determined using fungal importin-beta homolog Kap95, human Importin-beta•Ran(GTP) adopts a compacted superhelical pitch in vitrified environments captured by cryoEM, rather than an extended open orientation as previously observed. This configuration helps explain a poorly understood observation within the field, that Ran(GTP) bound importin-beta, despite also crossing the NPC to gain access to the cytoplasm, displays reduced affinity for FG-repeat motifs. In this study, reduced FG-repeat affinity upon binding Ran(GTP) is validated biochemically, with superhelical compaction observed in the structure proposed to close the deep clefts between HEAT repeats, shrinking the pocket size that FG motifs plunge into on the convex face of the transport factor. Overall this is a well thought out manuscript, that is clearly written and describes for the first time the depth of conformations required to conceptually understand cargo-recognition, transit, and release mechanics across a single beta-type karyopherin. It is my view that this approach represents a blueprint that will be invaluable in teasing out the mechanisms by which a comparatively small suite of nuclear transport factors (~17 in humans) can selectively translocate about eight thousand different nuclear cargos. Nevertheless, while I remain very enthusiastic about this manuscript it does require minor revisions, primarily graphical issues, which hold back the high quality of data presented and limit this papers appeal beyond the nucleocytoplasmic transport field.\nMajor comments:\n(1) Figure 1. The schematic representation of importin-beta mediated nuclear import across the NPC is rudimentary and lacks the required detail to orient a broad readership. For example, there are no arrows indicating directionality. Additionally, several of the introduced factors such as Importin-alpha1 and RanBP1 appear and disappear without reference to where they have come from, this is jarring as RanBP1 containing complexes are shown at the nuclear side of the NPC, yet the text correctly posits on lines 61 to 71, and lines 107 to 116 that RanBP1 is primarily cytoplasmic and plays a role in promoting Ran(GTP) hydrolysis in tandem with the Ran GTPase activating protein, Ran GTPase activating protein, RanGAP1. However based on this figure the Ran cycle from nuclear Ran(GTP) to cytoplasmic Ran(GDP), which is subsequently reimported by NTF2 is largely lost, making these details hard to follow for the general reader. In summary, this figure provides scant context for where the determined structures fit within the importin-beta nuclear import cycle and should be overhauled to encompass the entire cycle with the steps that have been structurally characterized in this study highlighted.\n(2) The presentation of the cryoEM data throughout the figures lacks standard representations such as local resolution and anisotropy across the structure, and flow diagrams that detail the number of movies/particles collected and the data processing steps taken to obtain the final reconstructions.\n(3) In line with the above comment a supplementary figure that highlights the quality of the electron density maps for each structure at topical or interesting regions would be appreciated.\n(4) Figure 3C. The FG binding pocket schematic does not encompass all five binding interfaces visualized in the structure. While the focus on the novel sites is understandable, a complete view would aid in broader comprehension.\n(5) Figure 6A. The chicken wire structural representation for the helical solenoid of importin-beta is passable in the earlier figures, however in the structural superposition becomes extremely challenging to interpret. A possible solution would be to either render these cartoons with cylindrical helices, or expand this analysis into the supplement where each structure could be compared in a pairwise fashion.\n(6) Figure 8. GST pulldowns probing importin-beta binding to various FG domains in the presence and absence of Ran(GTP). These pulldowns lack loading gels that indicate the relative loading levels of each protein relative to one another, additionally, the molecular weight marker is unlabeled.\n(7) Unbound importin-beta structure. Whilst there is no structure of the unbound human importin-beta a fungal homolog has been determined (Forwood et al., 2010 Structure), with the same study presenting SAXS data to support a similarly compressed conformation for the human importin-beta in solution. AlphaFold models of importin-beta in isolation appear to match this orientation (although this is meaningless as it is not experimentally validated), thus a comparison with your IBB cargo/Ran bound structures, or better yet a bonafide apo importin-beta structure determined by cryoEM should be addressed in the discussion section.\nMinor comments:\n(1) Line 49. “The small GTPase Ran, enriched as Ran-GTP in the nucleus and Ran-GDP in the cytoplasm, facilitates the import reaction by influencing the NPC permeability.” The cited paper (Lowe et al., 2015 eLife) indicates that NPC permeability is dictated by importin-beta accumulation within the NPCs central channel, please rework this sentence.\n(2) Figure 2B. The superposition of the IBB domains for importin-alpha1 and xRIP-alpha is welcome, however a more informative superposition would be the importin-beta solenoid which varies its superhelical pitch between these structures.\n(3) Figures 2-5. The density indicted for the molecules bound to importin-beta lacks sufficient contrast.\n(3) Line 281. “Ran-GTP reduces the affinity of importing-beta for FG-nups”, this statement requires a citation.\n(4) Figure 9B. The MBP-importin-alpha IBB titration demonstrates formation of a tetrameric intermediate wherein both Ran(GTP) and the cargo-IBB are bound to importin-beta concurrently. However, an equivalent titration following increasing [importin-alpha IBB] against preformed importin-beta•Ran(GTP) dimers would be useful to determine whether RanBP1 binding is a prerequisite for cargo-ran intermediate formation.\n(5) Table 1. One entry missing in the row “Chains / Residues.”\n(6) Methods section. More detail would be appreciated for the cloning, expression, and purification of recombinant proteins used in this study, for example what are the plasmids used where are the affinity purification tags located (N/C-termini), what expression conditions were used, whilst citing prior work is acceptable for protein purifications it should not be in lieu of a detailed description of the protocols used in the current manuscript.\n(7) Throughout the paper the functional localization of RanBP1 gets a little confusing, for example in lines 413 421 RanBP1 is proposed to aid in the translocation of the import complex through the NPC into the nucleoplasm, while I have no trouble envisioning RanBDs/ZnFs supplied by the nuclear basket nucleoporins Nup50 or Nup153 occupying this role, the proposition that cytoplasmic RanBP1 is involved in nucleoplasmic complex release does require justification.\n(8) A discussion of the recent single molecule NPC translocation data published in Sau et al., 2025 Nature, tracking both cargo-bound importin-beta from the cytoplasm and presumably Ran(GTP) bound nuclear export would be a nice addition when describing the FG pulldowns, for example does the observation that importin-beta binds FGs significantly weaker in one configuration tally with the measured nuclear export rates of importin-beta."}, "author_response": "Point-by-point response to reviewers’ critiques\nWe thank the reviewers and the editor for carefully reading our paper and offering helpful comments.\nWe have thoroughly revised the paper to incorporate all the reviewers' feedback and added new data.\nWe believe the revised version is significantly improved. Key changes in this manuscript include:\n1 – We solved a new conformation of importin  bound to Ran-GDP:RanBP1, presented in a new\nFigure 6. Overall, the revised paper features six cryo-EM reconstructions of human importin ,\nrepresenting five distinct conformations of the protein, each with unique biochemical activities. The new\nstructure of importin β broadens the scope of this paper and sheds light on the mysterious importin\nβ:Ran-GDP:RanBP1 complex, which many groups have studied over the last 30 years.\n2 – We measured the biochemical activity of the ternary importin :Ran-GDP:RanBP1 complex for FG-\nnups (revised Figs. 8 and 9), and for cargo (new Supplementary Fig. S10). Comparing the previously\ndescribed importin :Ran-GTP:RanBP1 complex with the new importin :Ran-GDP:RanBP1 complex\nprovides further insights into nuclear transport biology, which allowed us to expand the Discussion\n(pages 14-16). These new data thoroughly address questions raised by reviewers and enabled us to\nexpand the discussion in the revised paper.\n3 – We added a new Supplementary Figure S1 illustrating all the Fourier Shell Correlation curves and\nlocal resolution maps for the six cryo-EM reconstructions presented in this paper, as requested by Rev\n#2.\n4 – We added a new Supplementary Figure S2 showing representative features for the six cryo-EM\nreconstructions presented in this paper, as requested by Rev #2.\n5 – We expanded the analysis of the previously reported Kap95p:Ran-GTP complex (published in\nNature in 20041). In a new Supplementary Fig. 4, we illustrate how this crystal structure differs from\nthe closed conformation of human importin β observed by cryo-EM, due to crystallization-induced\ndimerization of Kap95p C-termini. Although this conformation is non-physiological, we show that\nKap95p FG-binding pockets are closed in the Kap95p:Ran-GTP crystal structure (shown in a new\nSupplementary Fig. 6), supporting our model of allosteric crosstalk across importin β surfaces\nmediated by Ran-GTP. These new figures directly respond to a suggestion made by Rev #1.\nOverall, we believe the revised paper is both broader in scope and more rigorous, ready to engage a\nwide audience of cell and structural biologists. Below, we provide a detailed, point-by-point response\nto all reviewer comments. Changes in the revised paper are highlighted in red.\nREVIEWER COMMENTS\nReviewer #1 (Major concerns)\nThe cryo-EM structures of the IBB–importin-β complexes appear substantially more elongated\ncompared to prior X-ray structures. Given the known flexibility of importin-β, the observed\nconformations may represent only a subset of possible configurations. Furthermore, the FG-repeat-\nbound importin-β structure appears structurally similar to the IBB-bound form. The authors report\nselecting ~4.3 million to ~11.9 million particles for initial processing across different complexes, but the\nfinal reconstructions are based on significantly fewer particles (e.g., 293,677 for importin-β–xIBB and\n1,400,279 for importin-β–Ran-GTP). This discrepancy raises concerns about particle heterogeneity and\nwhether the resolved structures represent physiologically dominant states.\n:FG complex was more aggregated because of a slight excess of nucleoporin over importin .\nTruthfully, it's impossible to compare particles used or rejected from two different datasets. In Single\nParticle Analysis (SPA), we retain 2D projections that can be aligned and used to generate a high-\nresolution reconstruction. The particles discarded during a SPA analysis workflow are usually broken,\nmisfolded, or adopt conformations that do not conform to the 2D-class averages. We wholeheartedly\nagree with the reviewer that importin  can sample more states in solution than those we were able to\nreconstruct by cryo-EM and present in this paper. However, assuming an unbiased distribution of\nparticles on a grid (as in your case), the 3D reconstructions presented in this paper represent the most\npopulated conformations that importin  adopts in solution in complex with its binding partners. Please\nnote that we were unable to reconstruct the apo-importin (without binding partners) because we\ncannot align the 2D projections of apo-importin . The unliganded protein fluctuates through many\nstates in solution 2,3 (all captured on ice), making 2D classification and, therefore, 3D reconstruction,\nimpossible.\nAs someone trained in crystallography and the first to successfully crystallize human importin β\nin the late 1990s, I must admit that cryo-EM SPA is not perfect. However, it marks a major advance\nover X-ray crystallography, where what we observe in the crystal is simply what we were able to\ncrystallize, often an artifact caused by artificial pH, super-physiological concentrations of protein, or\ncrystallization agents (which disrupt the structure of water). Even for a macromolecule that crystallizes\nin its native conformation, the conformation seen in a crystal lattice is often heavily influenced by lattice\nforces that strengthen, compress, and trap macromolecules in local minima.\nIt would be informative to assess whether the selected particles reflect a major or minor population and\nto discuss the implications for physiological relevance.\nAgreed! What the reviewer is asking for is every structural biologist's dream, which, unfortunately, is\ncurrently impossible to achieve. The particles selected for 3D reconstruction are those that can be\naligned with high confidence. The reviewer is correct that there is (a lot of) information we do not capture\nin a cryo-EM 3D reconstruction; this is a well-known and documented limitation of cryo-EM SPA. It also\nexplains why we must keep cryo-micrographs for many years. Future software will be able to examine\nconformational heterogeneity that we now discard. To fully address the reviewer’s point, we expanded\nthe Discussion section of the revised paper to clearly outline the limitations of cryo-EM SPA for flexible\nproteins, such as importin  (page 13, lines 477-488).\nIn addition, comparison of FG-repeat-bound importin-β structures obtained by both cryo-EM and X-ray\ncrystallography would strengthen the argument.\nDone! We have improved the comparison between our cryo-EM structure of importin :FG and the\ncrystal structure of Kap95p, solved in complex with Nup1 (new Supplementary Fig. 6A). We state in\nthe text that Kap95p FG-binding pockets #1, #2 and #3 seen in the crystal structure correspond to\nhuman importin ’s pockets #1, #2, and #4 observed by cryo-EM (page 10, lines 369-381). Additionally,\nwe validated our model of Ran-induced allosteric crosstalk between the importin  surfaces by\ncomparing Kap95p FG-pockets in the two previously reported crystal structures, PDBs 5OWU and\n2BKU (see below).\nThe cryo-EM structure of the RanGTP–importin-β complex reveals a closed configuration, particularly\nat the C-terminus, which differs from the open conformation observed in the Kap95p–RanGTP complex.\nIt is critical to determine whether crystal structures of RanGTP–importin-β also show reduced solvent\naccessibility of FG-binding pockets. Furthermore, since the decrease in solvent-accessible volume\n2\nupon RanGTP binding is a key finding, functional validation through mutagenesis, followed by cell-\nbased assays, is necessary to test the physiological impact of this structural change.\nDone! Thank you for pointing this out. In the revised paper, we have thoroughly re-analyzed the\npreviously published X-ray structures of Kap95p bound to Ran-GTP and Nup1 and fully validated our\nmodel for the allosteric opening of FG-pockets by Ran-GTP. Specifically, on pages 6, lines 213-223,\nand Supplementary Fig. 4, we clarify that Kap95p:Ran-GTP complex fortuitously crystallizes as a\ndimer, which – we believe – represents a crystallographic artifact. However, two of the FG-binding\npockets in Kap95p occupied by an FG in the crystal structure of Kap95p:Nup1 are closed in complex\nwith Ran-GTP, validating our model in full. This analysis is described in a new section of the revised\npaper, on page 10, lines 369-381, and illustrated in a new Supplementary Fig. 6.\nRegarding the reviewer’s request for additional functional validation and mutagenesis, importin\nFG-pockets have been well studied and mutated in previous research (discussed on lines 173-192,\npage 5). Our cryo-EM reconstruction confirms all prior mutagenesis studies of FG-pockets in importin\n and highlights weaker sites, as shown by molecular dynamics (MD) 4. We currently have no additional\nsites to validate through mutagenesis and cell-based assays. We aim to employ site-directed\nmutagenesis and cell-based assays to investigate residues that convey allosteric signals from the inner\nsurface of importin which binds Ran-GTP, to the FG-binding pockets on the outer surface. However,\nwe are not ready to undertake such an extensive effort at this stage. The distance between importin \nsurfaces spans dozens of Å, with hundreds of residues potentially involved in relaying allosteric signals.\nFirst, we need to perform equilibrium molecular dynamics simulations of importin  bound to Ran-GTP\nand IBB/FG-nup to identify and prioritize residues for mutagenesis. Only then can we introduce and\ntest specific mutations in importin Each importin variant will need to be tested for proper folding (as\nmutations at the HEAT:HEAT repeat interface can misfold importin ), binding to IBB/FG-nup and\nRan-GTP, and ultimately, probed for nuclear import in cell-based assays. We cannot directly move from\ncryo-EM structures to biochemical validation without an intermediate step, which only MD simulations\ncan currently provide. However, this would require years of work, with no guarantee of identifying a\nspecific residue involved in allostery, which can be transduced by biochemically undetectable small\nchanges distributed across many residues. Thus, with the editor’s approval, we will reserve this analysis\nfor future study.\nA major concern is the stability and physiological relevance of the RanBP1–importin-β–RanGTP\ncomplex. RanBP1 is known to facilitate the dissociation of RanGTP from importin-β, promoting complex\ndisassembly and recycling of importin-β. Therefore, the trimeric complex is expected to be transient\nand highly dynamic, not forming a stable assembly under physiological conditions. The complex\nobserved in vitro likely reflects an artificial state induced by experimental conditions. The authors should\ndiscuss this discrepancy and explicitly address the likely non-physiological nature of the RanBP1–\nimportin-β–RanGTP complex.\nAgree! The reviewer is correct that the trimeric importin β:Ran-GTP:RanBP1 complex is likely transient\nin a cell (when the Ran gradient is in place). However, this has not prevented researchers from\nidentifying and characterizing this trimeric species that is well-documented in the literature. At least four\nindependent groups have reported the identification and characterization of the importin-\nβ:RanGTP:RanBP1 (ternary) complex over the past 30 years. As early as 1997, the Macara lab 5\nshowed RanBP1 forms a ternary complex with Ran and importin β biochemically. Bischoff & Görlich 6\ndemonstrated RanBP1’s role in releasing RanGTP from import receptors and described RanBP1-\ncontaining receptor:Ran intermediates. Kutay et al. 7 detected importin β:Ran-GTP:RanBP1 assemblies\nin studies of dominant-negative importin β and transport pathways. Sarić et al. 8 determined a crystal\n3\nstructure and provided biochemical characterization of importin β (1-445) bound to Ran-GTP:RanBD\n(Ran-binding domain of RanBP1 lacking the N-terminal helix), further providing direct structural\nevidence for a stable ternary complex. In kinetic studies, Villa et al. 9 characterized the rate constant of\nthe importin  binding to RanBP1 and Ran-GTP, again characterizing a stable complex. All these\npapers are referenced in the revised paper.\nBut answering the reviewer’s comment more directly (“The authors should discuss this discrepancy\nand explicitly address the likely non-physiological nature of the RanBP1–importin-β–RanGTP\ncomplex”), we can trap a stable importin β:Ran-GTP:RanBP1 complex for cryo-EM studies because\nour Ran protein has a Q69L mutation that prevents GTP hydrolysis. This mutation stabilizes a trimeric\ncomplex poised for dissociation. We now explicitly state this concept on page 6, lines 229-230.\nTo expand on the reviewer’s comment, in the revised paper, we also investigated the structure and\nbiochemical activity of the importin β:Ran-GDP:RanBP1 complex, which was identified by many labs,\nboth in vitro 5,9-12 and in live cells 13, roughly at the same time as the importin β:Ran-GTP:RanBP1\ncomplex was discovered. We determined a cryo-EM structure of the importin β:Ran-GDP:RanBP1\ncomplex (new Figure 6) and probed the affinity of importin β:Ran-GDP:RanBP1 for FG-nups (revised\nFigs. 8 and 9) and IBB-domain (new Supplementary Fig. S10). These new data indirectly address\nsome of the concerns raised by the reviewer. Unlike the importin β:Ran-GTP:RanBP1 complex, which\nis poised for GTP hydrolysis and dissociation, the importin β:Ran-GDP:RanBP1 complex has Ran\nlocked in a GDP state, making it less transient, although it has remained mysterious in the literature.\nOur new data reveal unique structural and biochemical differences between the two importin β\ntrimeric species. We do not support a role for the importin β:Ran-GDP:RanBP1 complex in the\ntranslocation of NLS cargos through the NPC. Instead, we hypothesize that the importin α:NLS cargo\nmay be temporarily escorted by importin β:Ran-GTP:RanBP1 to prevent stalling at high-affinity FG-\nbinding sites inside the NPC (discussed in the revised Discussion, on pages 14-16).\nIn Fig. 8, it is shown that importin-β alone binds FG-Nups more strongly than cargo-bound importin-β.\nYes! My lab reported in 2010 14 that the affinity of importin  for Nup62 is influenced by the IBB domain,\nin vitro. Using surface plasmon resonance, we found that free importin  binds the full-length Nup62\nwith a Kd value of approximately 8.04 ± 0.2 nM; in the presence of the IBB domain, the affinity for\nNup62 was reduced by about 40-fold (Kd = roughly 335 ± 90 nM), and with the snurportin IBB sIBB\n(res. 1–65), importin  affinity for Nup62 dropped to 1.58 ± 0.4 μM: a 200-fold decrease in affinity\ncompared to the unliganded importin . This is now stated on page 11, lines 390-394.\nThe authors should clarify whether importin-β alone can shuttle through the nuclear pore independently.\nYes! Importin  can shuttle through the NPC on its own, without cargo or an adaptor. This was first\nshown by direct translocation assays in digitonin-permeabilized cells, where purified importin \naccumulates at NPCs and can enter the nucleus independently of importin  or cargo. Görlich et al 15\nreconstituted nuclear import and found that importin  alone interacts strongly with FG-nucleoporins\nand traverses the NPC. Follow-up studies showed that importin  constantly shuttles between the\ncytoplasm and nucleus, even without cargo 7. Single molecule/FRAP experiments 16 directly observed\nimportin  shuttling through NPCs, revealing transient interactions with FG repeats and a characteristic\ntransport dwell time. All this is now stated in the revised text on page 2, lines 55-59.\ninto the nuclear pore regulated?\nYes! The Weis lab reported in eLife that there are at least two pools of importin  within the NPC 17.\nOne pool is stably bound to the NPC for many minutes, regardless of the presence of Ran-GTP. The\nother pool is stably bound to the NPC only when Ran-GTP is absent. We speculate that the Ran-GTP-\nsensitive pool is the one that moves through the NPC. This remains an open question. We state this\npoint in the revised Introduction on page 2, lines 59-61.\nMinor issues:\n1. Figure 8 (SDS-PAGE): The SDS-PAGE gel in Figure 8 is inadequately labeled (e.g., molecular weight\nmarkers).\nFixed! Thank you for pointing this out. Because we performed an additional three sets of pull-downs\nwith Ran-GDP and Ran-GDP:RanBP1, the pull-down gels shown in the main text have been cropped\nto fit six pull-downs in Figure 9 (formerly Figure 8). We present the entire MW ladder and uncropped\ngels in a new Supplementary Figure 7.\n2. Importin-β Protein Disappearance in Pull-Down Assays (Figure 8):\nIn the pull-down assays using GST-tagged FG-Nups, importin-β protein seems to disappear in both the\nbound and unbound fractions (lanes 4–6 and 10–12). Moreover, a quantitative analysis plotting the\nratio of importin-β in bound fractions over total input should be provided to ensure consistent protein\nloading across samples.\nFixed! Thank you for pointing this out. We made a mistake in the submitted manuscript and apologize\nfor it. The fractions shown in lanes 9-12 are ‘Controls’, representing the purified proteins used for pull-\ndown, not ‘Unbound’ as labeled in the figure. We do NOT show unbound fractions in this experiment\nbecause there is too little protein to see clear bands by SDS-PAGE. Pull-downs (Fig. 9A-C) are\nperformed in 50 L GST-beads incubated with physiological concentrations of importin  (3 M) alone\nor importin  (3 M) together with 3 M IBB, 3 M Ran-GTP, or 3 M Ran-GTP plus 3 M RanBP1.\nSimilarly, in the revised paper, we have also added new pull-downs (Fig. 9D-F) using importin  (3 M)\nalone or importin  (3 M) together with 3 M IBB, 3 M Ran-GDP, or 3 M Ran-GDP plus 3 M\nRanBP1. The unbound fraction is too dilute to be visible with Coomassie staining, unlike the bound\nfraction that concentrates on the beads. This explains why lanes 2-6 of each gel are labeled as ‘Bound\nto GST-FG-nup’. Once again, we apologize for not explaining this clearly in the text. We have revised\nthe text (page 10-11, lines 384-412), methods (page 18, lines 672-691), and figure legend (page 29,\nlines 1,074-1,087) to clarify this point. We also include a new Supplementary Fig. 7, which shows the\nuncropped pull-down gels.\n3. GST-tagged Nup358 (residues 2503–2893), full-length Nup62, and Nup153 (residues 946–1472)\nshould have distinct molecular weights. However, they appear at comparable positions on the SDS-\nPAGE gel (Figure 8).\nAgree! The molecular weights of these GST-tagged fragments of nups are as follows:\nGST-Nup358 (res. 2503-2893): M.W. = 26 + 42.4 = 68.4 kDa;\nGST-Nup62 (res 1-522): M.W. = 26 + 62 = 88.0 kDa\nGST-Nup153 (res. 946-1472): M.W. = 26 + 58.7 = 84.7 kDa\nBecause of the small difference in M.W., it is not surprising that all three FG-nups migrate between\nthe 70 kDa and 100kDa molecular weight markers.\n4. Figure 9 (Native Gel and Quantification): In Figure 9, if IBB binds to the trimeric complex (importin-\nβ–RanGTP–RanBP1), the intensity of the trimeric complex should decrease.\nHowever, this reduction is not evident.\nThe authors should provide a quantitative\ndensitometry analysis to support their\ninterpretation.\nDone! We quantified the band shift data in\nresponse to the reviewer’s question. To do\nso, we used a less-stained version of the\nsame gel shown in Fig. R1A, which we\ntook before submitting the paper (e.g., the\ngel in the submitted paper was over-\nstained to increase contrast). The relative\nquantification of bands is shown in Fig.\nR1B). We observed an apparent decrease\nin the intensity of the importin :Ran-\nGTP:RanBP1 band, which drops by about\n50% in the presence of 8x to 10x IBB.\nAlong with the disappearance of the\ntetrameric importin :Ran-GTP:RanBP1\ncomplex, we also observed a concomitant\nappearance of the tetrameric importin\n:Ran-GTP: RanBP1:IBB band (black\nhistogram in Fig. R1B). Granted, the trend\nis evident; however, we consider native Figure R1. Quantification of band intensities for lanes 4\ngels stained with Coomassie to be ([IBB] = 0x) through 12 ([IBB] = 10x) of the native page. (A)\nintrinsically qualitative and therefore Under-stained native gel, same as that presented in Fig. 10B. (B)\nunsuitable for rigorous quantification with Quantification of the trimeric importin :Ran-GTP:RanBP1\nstatistical analysis and standard deviation. complex (gray), and tetrameric importin :Ran-GTP:RanBP1:IBB\nProtein migration under native conditions complex (black). In panel (B), the Y axis (normalized to 100%) is\nis subject to numerous sources of the concentration of importin :Ran-GTP:RanBP1:IBB complex\nsystematic error—significantly more fixed in the titration. The loss of importin :Ran-GTP:RanBP1 band\nproblematic than in classical SDS– intensity is about 50% when the concentration of IBB is between\nPAGE—including sample loss during 8x (lane 11) and 10x (lane 12).\nloading, band smearing, and uneven staining. Therefore, we will not include this data in the revised\npaper. We added the statement on page 12, lines 443-445: ‘Quantification of native gel bands indicated\nthat roughly half of the trimeric importin :Ran-GTP:RanBP1 complex was converted into a tetrameric\ncomplex upon addition of a 5–10-fold molar excess of IBB (data not shown)’.\n4. Interpretation of Figure 9B: The conclusion that \"association of RanBP1 with importin-β and RanGTP\npromotes binding to αIBB\" is unclear, especially since RanGTP typically promotes dissociation of\nimportin-α/β–cargo complexes. A more detailed mechanistic explanation is required to reconcile these\nfindings.\nabsence of RanBP1, IBB cannot associate with the importin :Ran-GTP heterodimer. Therefore, it is\nthe binding of RanBP1 to importin β and Ran-GTP that stabilizes a conformation of the importin  C-\nterminus compatible with IBB association. This is now described on page 12, lines 445-447.\nFurthermore, also relevant to the reviewer’s point, we added a new Supplementary Fig. 10 that\ndemonstrates that, unlike the Ran-GTP:RanBP1 complex, importin  associated with Ran-\nGDP:RanBP1 fails to bind the IBB. This new data demonstrate that the nucleotide-bond state of Ran\ndetermines importin ’s ability to associate with cargo and FG-nups in a RanBP1-dependent manner\n(described on page 12, lines 451-458).\n5. Validation of FG-Nup Displacement by αIBB Binding (Competition Assay):\nIn the Discussion, the authors propose that a transient importin-β:α1:NLS-cargo complex, when\nsimultaneously bound to RanGTP and RanBP1, exhibits reduced avidity for FG-Nups, facilitating\npassage through the NPC. However, the current experiments primarily demonstrate that αIBB can bind\nto the RanGTP–RanBP1–importin-β complex. To directly test the physiological relevance of the model,\nit is important to perform a competition assay to determine whether αIBB binding actively displaces pre-\nbound FG-Nups from importin-β. Such an experiment would provide more definitive evidence\nsupporting the authors’ proposed mechanism.\nAgreed! We have previously reported 14 that the IBB-domain is not sufficient to displace FG-nups from\nimportin , but its association with importin  reduced the binding affinity of Nup62 by 40-fold. A more\nsubstantial effect was observed for sIBB that reduces importin ’s affinity for Nup62 by 200-fold (See\nFig. R2). Thus, the IBB can weaken the association of importin  for FG-nups but is not sufficient to\nactively displace nucleoporins. This is now stated in the revised manuscript on page 11, lines 390-392.\nA B C\nImp β Imp β:IBB Imp β:sIBB(1-65)\n100 500\n300\nU) U) \nResponse\nUnits ( R\n2\n4\n6\n0\n0\n0\n0\n0\n0\n0\n.\n.\n.\n.\n2\n1\n1\n5\n0\n0\n5\n0\nµ\nµ\nµ\nM\nM\nM\nResponse\nUnits ( R\n1\n2\n3\n0\n0\n0\n0\n0\n0\n2\n3\n4\n5 µ\nµ\nµ\nM\nM\nM\nResponse\nUnits ( R U)\n1\n2\n0\n0\n0\n0 1\n6\n9\n2\nµ\nµ M\nM\n0.05 µM 3\n1 µM\n0 0 1 µM\n1200 1350 1500 1650 1800 1950 1200 1350 1500 1650 1800 1950 0\n1200 1350 1500 1650 1800 1950\nTime (s) Time (s)\nTime (s)\nFigure R2. The IBB-domain decreases the affinity of importin  for Nup62. Surface Plasmon Resonance (SPR)\nanalysis of the interaction of importin β alone (A) or bound to αIBB (B), or sIBB(1-65) with Nup62 captured onto a\nsensor chip. In each sensogram, raw data are shown as open circles and fit curves as black lines for five different\nconcentrations. A representative Kd for each interaction is shown next to the relative sensogram. Adapted from Lott et\nal., J Biol Chem, 2010.\nReviewer #2 (Major comments):\n(1) Figure 1. The schematic representation of importin-beta mediated nuclear import across the NPC\nis rudimentary and lacks the required detail to orient a broad readership. For example, there are no\narrows indicating directionality. Additionally, several of the introduced factors such as Importin-alpha1\nand RanBP1 appear and disappear without reference to where they have come from, this is jarring as\nRanBP1 containing complexes are shown at the nuclear side of the NPC, yet the text correctly posits\non lines 61 to 71, and lines 107 to 116 that RanBP1 is primarily cytoplasmic and plays a role in\n7\npromoting Ran(GTP) hydrolysis in tandem with the Ran GTPase activating protein, RanGAP1.\nHowever based on this figure the Ran cycle from nuclear Ran(GTP) to cytoplasmic Ran(GDP), which\nis subsequently reimported by NTF2 is largely lost, making these details hard to follow for the general\nreader. In summary, this figure provides scant context for where the determined structures fit within the\nimportin-beta nuclear import cycle and should be overhauled to encompass the entire cycle with the\nsteps that have been structurally characterized in this study highlighted.\nDone! Thank you for the feedback. We have updated Fig. 1 to be more comprehensive.\n(2) The presentation of the cryoEM data throughout the figures lacks standard representations such as\nlocal resolution and anisotropy across the structure, and flow diagrams that detail the number of\nmovies/particles collected and the data processing steps taken to obtain the final reconstructions.\nDone! We have added a new Supplementary Fig. 1, which includes Fourier Shell Correlation curves\nand local resolution maps. We also included the number of movies/particles in Table 1.\n(3) In line with the above comment a supplementary figure that highlights the quality of the electron\ndensity maps for each structure at topical or interesting regions would be appreciated.\nDone! We added representative high-resolution density features for all the cryo-EM reconstructions in\nthe paper to a new Supplementary Fig. 2.\n(4) Figure 3C. The FG binding pocket schematic does not encompass all five binding interfaces\nvisualized in the structure. While the focus on the novel sites is understandable, a complete view would\naid in broader comprehension.\nDone! Thank you for pointing this out. In the revised paper, we have included schematics for the\nremaining FG-binding pockets not shown in Figure 3C (e.g., pocket #2 and #3) in a new\nSupplementary Fig. 5.\n(5) Figure 6A. The chicken wire structural representation for the helical solenoid of importin-beta is\npassable in the earlier figures, however in the structural superposition becomes extremely challenging\nto interpret. A possible solution would be to either render these cartoons with cylindrical helices, or\nexpand this analysis into the supplement where each structure could be compared in a pairwise fashion.\nDone! We replaced Fig. 6A (now Fig. 7A) with a more minimalistic and possibly informative beads-on-\na-string representation of all importin  solenoids visualized in this paper. We kept panel B as (thick)\nchicken wire to match the individual reconstructions shown in Figs. 2-6.\n(6) Figure 8. GST pulldowns probing importin-beta binding to various FG domains in the presence and\nabsence of Ran(GTP). These pulldowns lack loading gels that indicate the relative loading levels of\neach protein relative to one another, additionally, the molecular weight marker is unlabeled.\nFixed! Thank you for pointing this out. As stated in response to Rev #1’s comments, we made a mistake\nin the submitted manuscript and sincerely apologize for this. The fractions shown in lanes 9-12 are\n‘Controls’, not ‘UnBound’, as labeled in the figure. We do NOT show unbound fractions in this\nexperiment, as there is too little protein to see clear bands by Coomassie staining. We revised this\nfigure to include six sets of pull-downs for three FG-nups probed with importin  in the presence of Ran-\nGTP (Fig. 9, panels A-C) and Ran-GDP (Fig. 9, panels D-F). We have revised the text (page 10-11,\nlines 383-412), methods (page 18, lines 672-690), and figure legend (page 29, lines 1,074-1,087) to\nclarify this point. We also include a new Supplementary Fig. 7 with the uncropped pull-down gels.\n(7) Unbound importin-beta structure. Whilst there is no structure of the unbound human importin-beta\na fungal homolog has been determined (Forwood et al., 2010 Structure), with the same study\npresenting SAXS data to support a similarly compressed conformation for the human importin-beta in\nsolution. AlphaFold models of importin-beta in isolation appear to match this orientation (although this\nis meaningless as it is not experimentally validated), thus a comparison with your IBB cargo/Ran bound\nstructures, or better yet a bonafide apo importin-beta structure determined by cryoEM should be\naddressed in the discussion section.\nThis is an excellent suggestion. We tried extensively to reconstruct the unliganded importin , but we\nare unable to ‘see’ the protein, which likely samples different conformations in solution (and on ice).\nThis makes it impossible to align using conventional single-particle analysis. Discuss in the discussion\non page 13, lines 477-488. We have collected SEC-SAXS data for the unliganded importin , but we\nhave not yet analyzed its structure. We will reserve this analysis for future study.\nMinor comments:\n(1) Line 49. “The small GTPase Ran, enriched as Ran-GTP in the nucleus and Ran-GDP in the\ncytoplasm, facilitates the import reaction by influencing the NPC permeability.” The cited paper (Lowe\net al., 2015 eLife) indicates that NPC permeability is dictated by importin-beta accumulation within the\nNPCs central channel, please rework this sentence.\nDone! Thank you very much for the feedback, and apologies for the incorrect statement. Ran regulates\nthe interaction between transport receptors (such as importin-β) and cargo, controlling the directionality\nof transport. NPC permeability is primarily governed by FG-nucleoporins, rather than by Ran. We\nrephrased the text to (page 2, lines 48-50): “The small GTPase Ran, enriched as Ran-GTP in the\nnucleus and Ran-GDP in the cytoplasm 18,19, facilitates the import reaction by regulating the interaction\nbetween transport receptors (like importin β) and cargo, controlling the directionality of transport 15”.\nWe also replaced the previous reference (Lowe et al) with Dirk Gorlich’s landmark paper on the role of\nRanGTP 15.\n(2) Figure 2B. The superposition of the IBB domains for importin-alpha1 and xRIP-alpha is welcome,\nhowever a more informative superposition would be the importin-beta solenoid which varies its\nsuperhelical pitch between these structures.\nDone! We provide this comparison in a new Fig. 2B.\n(3) Figures 2-5. The density indicted for the molecules bound to importin-beta lacks sufficient contrast.\nDone! We improved the contrast in the revised paper, which was also lost during the conversion to\nPDF. Representative densities for all binding partners visualized in complex with importin  are now\nincluded in a new Supplementary Fig. 2.\n9\n(3) Line 281. “Ran-GTP reduces the affinity of importing-beta for FG-nups”, this statement requires a\ncitation.\nDone! We now reference the seminal paper by Görlich et al. published in 1996 in EMBO J. 15.\n(4) Figure 9B. The MBP-importin-alpha IBB titration demonstrates formation of a tetrameric\nintermediate wherein both Ran(GTP) and the cargo-IBB are bound to importin-beta concurrently.\nHowever, an equivalent titration following increasing [importin-alpha IBB] against preformed importin-\nbeta•Ran(GTP) dimers would be useful to determine whether RanBP1 binding is a prerequisite for\ncargo-ran intermediate formation.\nDone! Thank you for pointing this out. We have performed the titration suggested by the reviewer and\nadded an increasing concentration of IBB to a preformed importin :Ran-GTP complex. As expected,\nIBB does not associate with importin  without RanBP1. These new data are now shown in a new\nSupplementary Fig. 9 and discussed on page 12, lines 445-450.\n(5) Table 1. One entry missing in the row “Chains / Residues.”\nFixed! We apologize for any shortcomings.\n(6) Methods section. More detail would be appreciated for the cloning, expression, and purification of\nrecombinant proteins used in this study, for example what are the plasmids used where are the affinity\npurification tags located (N/C-termini), what expression conditions were used, whilst citing prior work is\nacceptable for protein purifications it should not be in lieu of a detailed description of the protocols used\nin the current manuscript.\nDone! We provide more details about cloning vectors, affinity tags, purification, etc.\n(7) Throughout the paper the functional localization of RanBP1 gets a little confusing, for example in\nlines 413 421 RanBP1 is proposed to aid in the translocation of the import complex through the NPC\ninto the nucleoplasm, while I have no trouble envisioning RanBDs/ZnFs supplied by the nuclear basket\nnucleoporins Nup50 or Nup153 occupying this role, the proposition that cytoplasmic RanBP1 is\ninvolved in nucleoplasmic complex release does require justification.\nYes! The reviewer makes a good point. In the revised paper, we expanded the characterization of\nRanBP1 (and RanBP1-like domains) by studying the importin :Ran-GDP:RanBP1 complex, which was\nreported by several labs as early as the mid-90s. Our data reveal notable differences between these\ntwo ternary complexes (see new Figure 6 and revised Figs. 8, 9, and Supplementary Fig. S9).\nImportantly, our new data do not support a role for the importin :Ran-GDP:RanBP1 complex in the\ntranslocation of the import complex. We expanded the discussion of our paper to clarify the functional\nand structural differences between importin :Ran-GDP:RanBP1 and importin :Ran-GTP:RanBP1.\nSee the revised Discussion (pages 14-16). Overall, the new data support our hypothesis that the\ncomplex with RanGTP helps facilitate the passage of the import complex through the NPC by reducing\nthe affinity of importin for FGs.\n10\n(8) A discussion of the recent single molecule NPC translocation data published in Sau et al., 2025\nNature, tracking both cargo-bound importin-beta from the cytoplasm and presumably Ran(GTP) bound\nnuclear export would be a nice addition when describing the FG pulldowns, for example does the\nobservation that importin-beta binds FGs significantly weaker in one configuration tally with the\nmeasured nuclear export rates of importin-beta.\nYes! Thank you for bringing this excellent paper 20 to our attention. This work is in perfect agreement\nwith our model, which suggests that Ran-GTP and RanBP1 facilitate the displacement of importin \nfrom FG-nups. We now reference this paper in the Introduction (on page 2, lines 71-74) and in the\nDiscussion, on page 15, lines 560-566.\ndissociation by RanGTP. Nature 435, 693-696 (2005). https://doi.org/10.1038/nature03578\nmolecular spring. Structure 16, 906-915 (2008). https://doi.org/10.1016/j.str.2008.03.007\nsolenoid protein structures. Structure 18, 1171-1183 (2010).\nhttps://doi.org/10.1016/j.str.2010.06.015\nbeta. Structure 13, 1869-1879 (2005). https://doi.org/10.1016/j.str.2005.09.007\nwith p97 nuclear protein import. J Cell Biol 135, 559-569 (1996).\nhttps://doi.org/10.1083/jcb.135.3.559\nrelated nuclear transport factors. FEBS Lett 419, 249-254 (1997). https://doi.org/10.1016/s0014-\n5793(97)01467-1\nof importin-beta block multiple pathways of import and export through the nuclear pore complex.\nEMBO J 16, 1153-1163 (1997). https://doi.org/10.1093/emboj/16.6.1153\nbeta.Ran.GTP.RanBD1 complex. FEBS Lett 581, 1369-1376 (2007).\nhttps://doi.org/10.1016/j.febslet.2007.02.067\nand kinetic requirements for the interaction of Ran with the Ran-binding domains from RanBP2\nand importin-beta. Biochemistry 39, 11629-11639 (2000). https://doi.org/10.1021/bi001010f\nGDP/RanBP1 on nuclear import factor p97. J Biol Chem 272, 6818-6822 (1997).\nhttps://doi.org/10.1074/jbc.272.10.6818\nRan and karyopherin beta and reduces Ran GTPase-activating protein (RanGAP) inhibition by\nkaryopherin beta. J Biol Chem 272, 551-555 (1997). https://doi.org/10.1074/jbc.272.1.551\npromote the interaction of Ran with p97/beta-karyopherin, linking the docking and translocation\nsteps of nuclear import. J Biol Chem 271, 2357-2360 (1996).\nhttps://doi.org/10.1074/jbc.271.5.2357\nconformational changes and a Ran x GDP-importin-beta -RanBP1 complex in vitro and in intact\ncells. J Biol Chem 277, 30121-30127 (2002). https://doi.org/10.1074/jbc.M203006200\nmodulates the avidity of importin beta for the nuclear pore complex. J Biol Chem 285, 13769-\n13780 (2010). https://doi.org/M109.095760 [pii]\n10.1074/jbc.M109.095760\nRanGDP and RanGTP in nuclear protein import. EMBO J 15, 5584-5594 (1996).\nalpha/cargo complex dissociation at the nuclear pore. Proc Natl Acad Sci U S A 105, 8613-8618\n(2008). https://doi.org/10.1073/pnas.0710867105\nRan-dependent manner. Elife 4 (2015). https://doi.org/10.7554/eLife.04052\n12\nXenopus egg extracts. Science 295, 2452-2456 (2002).\nhttps://doi.org/10.1126/science.1068798\nof the constituents of the Ran system is essential for transport into and out of the nucleus. EMBO\nJ 16, 6535-6547 (1997). https://doi.org/10.1093/emboj/16.21.6535\nNature 640, 821-827 (2025). https://doi.org/10.1038/s41586-025-08738-0\n13"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have substantially strengthened the revised manuscript. They determined a new cryo-EM structure of importin-β bound to Ran-GDP:RanBP1, compared their cryo-EM structures to other X-ray structures, and performed additional pulldown assays, directly addressing the major concerns raised in my previous review. Moreover, they have clarified several conceptual issues in the revised text, which improves readability and helps the community better appreciate the significance of their findings. I therefore recommend publication of this article in Nature Communications.\nOne remaining concern is the quantification of complex stoichiometry. Although Dr. Cingolani clearly stated in the response letter that native gels stained with Coomassie are intrinsically qualitative and not suitable for rigorous statistical analysis, I remain uncomfortable with the revised manuscript’s statement that approximately half of the trimeric complex converts into a tetramer. To strengthen this claim, a quantitative plot—derived from alternative biochemical assays or at least semi-quantitative image analysis—would be highly recommended.", "reviewer_2": "Ko and colleagues are to be congratulated on their well-considered and impactful revisions, which significantly strengthen an already impressive manuscript. The new Importin-beta1•Ran(GDP)•RanBP1 structure rounds out the structural landscape, with 6 high-quality cryoEM reconstructions presented in total. Capturing the near-complete conformational landscape accessible to this prototypical import karyopherin. Importantly, these structural advances are complemented by enhanced biochemical data expanding on the relative strength of FG-binding events sampling all the presented cargo bound/release factor configurations, and thus the entire transport cycle, adding welcome mechanistic depth. Improved cryoEM data presentation, incorporating FSC-curves, local resolution maps, and representative density features, enhances an already rigorous study. Moreover, expanded discussion of both contemporary and foundational literature provides both clearer context and accessibility for readers outside the field, and will undoubtably boost the impact of this manuscript.\nFinally, the figures themselves have also benefitted from notable aesthetic refinements, improving their clarity across the board. Taken together, this revised manuscript represents a substantial improvement and is now ready for publication in Nature Communications without further delay. Congrats again to the authors."}, "author_response": "REVIEWER COMMENTS\nReviewer #1\nOne remaining concern is the quantification of complex stoichiometry. Although Dr. Cingolani clearly\nstated in the response letter that native gels stained with Coomassie are intrinsically qualitative and not\nsuitable for rigorous statistical analysis, I remain uncomfortable with the revised manuscript’s statement\nthat approximately half of the trimeric complex converts into a tetramer. To strengthen this claim, a\nquantitative plot—derived from alternative biochemical assays or at least semi-quantitative image\nanalysis—would be highly recommended.\nWe have now quantified the native gel shown in Figure 10B. This semi-quantitative image analysis is\nillustrated in Supplementary Fig. 10A and described in the corresponding figure l e gend. The native\ngel electrophoresis in Figure 10B was repeated three times, and the average band intensities were\nquantified densitometrically. Although the error bars are larger than we would prefer, the data confirm\nour statement that about half of the trimeric importin :Ran-GTP:RanBP1 complex converts to a\nquaternary importin :Ran-GTP:RanBP1:IBB complex in the presence of a 4-fold molar excess of\nIBB. We added this statement to the text (line 444):\n“Quantification of native gel bands indicated that about half (e.g., 52.1 ± 12.8%) of the trimeric\nimportin :Ran-GTP:RanBP1 complex was converted into a tetrameric complex upon addition\nof a 4-fold molar excess of IBB (Supplementary Fig. 10A)”.\nThis is the best possible outcome with native gels. All gels are provided in the Data Source file, and\nquantifications are detailed in the legend of Supplementary Fig. 10A."}]

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