GLFG and FxFG nucleoporins bind to overlapping sites on importin-beta

The interaction between nuclear pore proteins (nucleoporins) and transport factors is crucial for the translocation of macromolecules through nuclear pores. Many nucleoporins contain FG sequence repeats, and previous studies have demonstrated interactions between repeats containing FxFG or GLFG cores and transport factors. The crystal structure of residues 1-442 of importin-beta bound to a GLFG peptide indicates that this repeat core binds to the same primary site as FxFG cores. Importin-beta-I178D shows reduced binding to both FxFG and GLFG repeats, consistent with both binding to an overlapping site in the hydrophobic groove between the A-helices of HEAT repeats 5 and 6. Moreover, FxFG repeats can displace importin-beta or its S. cerevisiae homologue, Kap95, bound to GLFG repeats. Addition of soluble GLFG repeats decreases the rate of nuclear protein import in digitonin-permeabilized HeLa cells, indicating that this interaction has a role in the translocation of carrier-cargo complexes through nuclear pores. The binding of GLFG and FxFG repeats to overlapping sites on importin-beta indicates that functional differences between different repeats probably arise from differences in their spatial organization.     

The adoption of a twisted structure of importin-beta is essential for the protein-protein interaction required for nuclear transport

Importin-beta is a nuclear transport factor which mediates the nuclear import of various nuclear proteins. The N-terminal 1-449 residue fragment of mouse importin-beta (impbeta449) possesses the ability to bidirectionally translocate through the nuclear pore complex (NPC), and to bind RanGTP. The structure of the uncomplexed form of impbeta449 has been solved at a 2.6 A resolution by X-ray crystallography. It consists of ten copies of the tandemly arrayed HEAT repeat and exhibits conformational flexibility which is involved in protein-protein interaction for nuclear transport. The overall conformation of the HEAT repeats shows that a twisted motion produces a significantly varied superhelical architecture from the previously reported structure of RanGTP-bound importin-beta. These conformational changes appear to be the sum of small conformational changes throughout the polypeptide. Such a flexibility, which resides in the stacked HEAT repeats, is essential for interaction with RanGTP or with NPCs. Furthermore, it was found that impbeta449 has a structural similarity with another nuclear migrating protein, namely beta-catenin, which is composed of another type of helix-repeated structure of ARM repeat. Interestingly, the essential regions for NPC translocation for both importin-beta and beta-catenin are spatially well overlapped with one another. This strongly indicates the importance of helix stacking of the HEAT or ARM repeats for NPC-passage.     

Structural basis for the high-affinity binding of nucleoporin Nup1p to the Saccharomyces cerevisiae importin-beta homologue, Kap95p

Macromolecules are transported across the nuclear envelope most frequently by karyopherin/importin-beta superfamily members that are constructed from HEAT repeats. Transport of Kap95p (yeast importin-beta), the principal carrier for protein import, through nuclear pore complexes is facilitated by interactions with nucleoporins containing FG repeats. However, Nup1p interacts more strongly with Kap95p than other FG-nucleoporins. To establish the basis of this increased affinity, we determined the structure of Kap95p complexed with Nup1p residues 963-1076 that contain the high-affinity Kap95p binding site. Nup1p binds Kap95p at three sites between the outer A-helices of HEAT repeats 5, 6, 7 and 8. At each site, phenylalanine residues from Nup1p are buried in hydrophobic depressions between adjacent HEAT repeats. Although the Nup1p and generic FG-nucleoporin binding sites on Kap95p overlap, Nup1p binding differs markedly and has contributions from additional hydrophobic residues, together with interactions generated by the intimate contact of the linker between Nup1 residues 977-987 with Kap95p. The length and composition of this linker is crucial and suggests how differences in affinity for Kap95p both between and within FG-nucleoporins arise.     

Crystallization and initial X-ray diffraction characterization of complexes of FxFG nucleoporin repeats with nuclear transport factors

NTF2 and importin-beta are transport factors that mediate nuclear protein import and which interact with nuclear pore proteins (nucleoporins) during translocation from the cytoplasm to the nucleus through nuclear pore complexes. We employed a native gel electrophoresis method to assess the interaction of nucleoporin constructs that contain FxFG sequence repeats with NTF2 and truncation mutants of importin-beta to determine suitable fragments for crystallization. Based on these data, we obtained crystals of complexes between yeast NTF2 and a construct containing five FxFG nucleoporin repeats from the yeast nucleoporin Nsp1p and between a construct containing residues 1-442 of human importin-beta and the same nucleoporin construct. The yeast NTF2-nucleoporin crystals have trigonal symmetry and diffract past 2.8 A resolution using synchrotron radiation, whereas the importin-beta-nucleoporin complex crystals have P2(1)2(1)2 orthorhombic symmetry and diffract past 3.2 A resolution.     

Electrostatic interactions involving the extreme C terminus of nuclear export factor CRM1 modulate its affinity for cargo

The toroid-shaped nuclear protein export factor CRM1 is constructed from 21 tandem HEAT repeats, each of which contains an inner (B) and outer (A) α-helix joined by loops. Proteins targeted for export have a nuclear export signal (NES) that binds between the A-helices of HEAT repeats 11 and 12 on the outer surface of CRM1. RanGTP binding increases the affinity of CRM1 for NESs. In the absence of RanGTP, the CRM1 C-terminal helix, together with the HEAT repeat 9 loop, modulates its affinity for NESs. Here we show that there is an electrostatic interaction between acidic residues at the extreme distal tip of the C-terminal helix and basic residues on the HEAT repeat 12 B-helix that lies on the inner surface of CRM1 beneath the NES binding site. Small angle x-ray scattering indicates that the increased affinity for NESs generated by mutations in the C-terminal helix is not associated with large scale changes in CRM1 conformation, consistent with the modulation of NES affinity being mediated by a local change in CRM1 near the NES binding site. These data also suggest that in the absence of RanGTP, the C-terminal helix lies across the CRM1 toroid in a position similar to that seen in the CRM1-Snurportin crystal structure. By creating local changes that stabilize the NES binding site in its closed conformation and thereby reducing the affinity of CRM1 for NESs, the C-terminal helix and HEAT 9 loop facilitate release of NES-containing cargo in the cytoplasm and also inhibit their return to the nucleus.     

An allosteric mechanism to displace nuclear export cargo from CRM1 and RanGTP by RanBP1

The karyopherin CRM1 mediates nuclear export of proteins and ribonucleoproteins bearing a leucine‐rich nuclear export signal (NES). To elucidate the precise mechanism by which NES‐cargos are dissociated from CRM1 in the cytoplasm, which is important for transport directionality, we determined a 2.0‐Å resolution crystal structure of yeast CRM1:RanBP1:RanGTP complex, an intermediate in the disassembly of the CRM1 nuclear export complex. The structure shows that on association of Ran‐binding domain (RanBD) of RanBP1 with CRM1:NES‐cargo:RanGTP complex, RanBD and the C‐terminal acidic tail of Ran induce a large movement of the intra‐HEAT9 loop of CRM1. The loop moves to the CRM1 inner surface immediately behind the NES‐binding site and causes conformational rearrangements in HEAT repeats 11 and 12 so that the hydrophobic NES‐binding cleft on the CRM1 outer surface closes, squeezing out the NES‐cargo. This allosteric mechanism accelerates dissociation of NES by over two orders of magnitude. Structure‐based mutagenesis indicated that the HEAT9 loop also functions as an allosteric autoinhibitor to stabilize CRM1 in a conformation that is unable to bind NES‐cargo in the absence of RanGTP.     

Using peptide arrays to define nuclear carrier binding sites on nucleoporins

In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta.     

Conformational heterogeneity of karyopherin beta2 is segmental

Karyopherinbeta2 (Kap beta2) or transportin imports numerous RNA binding proteins into the nucleus. Kap beta2 binds substrates in the cytoplasm and targets them through the nuclear pore complex, where RanGTP dissociates them in the nucleus. Here we report the 3.0 A crystal structure of unliganded Kap beta2, which consists of a superhelix of 20 HEAT repeats. Together with previously reported structures of NLS and Ran complexes, this structure provides understanding of conformational heterogeneity that accompanies ligand binding. The Kap beta2 superhelix is divided into three major segments. Two of them (HEAT repeats 9-13 and 14-18), which constitute the substrate binding site, are rigid elements that rotate relative to each other about a flexible hinge. The third (HEAT repeats 1-8), which constitutes the Ran binding site, exhibits conformational changes throughout its length. An analogous segmental architecture is also observed in Importin beta, suggesting that it is functionally significant and may be conserved in other import karyopherins.     

A 2.1-Å-Resolution Crystal Structure of Unliganded CRM1 Reveals the Mechanism of Autoinhibition

CRM1 mediates nuclear export of numerous proteins and ribonucleoproteins containing a leucine-rich nuclear export signal (NES). Binding of RanGTP to CRM1 in the nucleus stabilizes cargo association with CRM1, and vice versa, but the mechanism underlying the positive cooperativity in RanGTP and NES binding to CRM1 remains incompletely understood. Herein we report a 2.1-Å-resolution crystal structure of unliganded Saccharomyces cerevisiae CRM1 (Xpo1p) that demonstrates that an internal loop of CRM1 (referred to as HEAT9 loop) is primarily responsible for maintaining the NES-binding cleft in a closed conformation, rendering CRM1 incapable of NES binding in the absence of RanGTP. The structure also shows that the C-terminal tail of CRM1 stabilizes the autoinhibitory conformation of the HEAT9 loop and thereby reinforces autoinhibition. Comparison with the structures of CRM1–NES–RanGTP complexes reveals how binding of RanGTP is associated with a series of allosteric conformational changes in CRM1 that lead to opening of the NES-binding cleft, allowing for stable binding of NES cargoes.     

Karyopherin flexibility in nucleocytoplasmic transport

Recent structural work on nuclear transport factors of the importin-beta superfamily of karyopherins has shown that these proteins are superhelices of HEAT repeats that are able to assume different conformations in different functional states. The inherent flexibility of these helicoids facilitates the accommodation of different binding partners by an induced-fit type of mechanism. Moreover, the energy stored by distorting these molecules may partially balance binding energies to enable assembly and disassembly of their complexes with relatively small energy changes. Flexibility appears to be an intrinsic feature of such superhelices and might be functionally important not only for karyopherins and nuclear transport, but also for HEAT repeat proteins from other biological systems.     

Role of importin-beta in the control of nuclear envelope assembly by Ran

Compartmentalization of the genetic material into a nucleus bounded by a nuclear envelope (NE) is the hallmark of a eukaryotic cell. The control of NE assembly is poorly understood, but in a cell-free system made from Xenopus eggs, NE assembly involves the small GTPase Ran. In this system, Sepharose beads coated with Ran induce the formation of functional NEs in the absence of chromatin. Here, we show that importin-beta, an effector of Ran involved in nucleocytoplasmic transport and mitotic spindle assembly, is required for NE assembly induced by Ran. Concentration of importin-beta on beads is sufficient to induce NE assembly in Xenopus egg extracts. The function of importin-beta in NE assembly is disrupted by a mutation that decreases affinity for nucleoporins containing FxFG repeats. By contrast, a truncated protein that cannot interact with importin-alpha is functional. Thus, importin-beta functions in NE assembly by recruiting FxFG nucleoporins rather than by interaction through importin-alpha with karyophilic proteins carrying classical nuclear localization signals. Importin-beta links NE assembly, mitotic spindle assembly, and nucleocytoplasmic transport to regulation by Ran and may coordinate these processes during cell division.     

Dominant-negative mutants of importin-beta block multiple pathways of import and export through the nuclear pore complex.

Nuclear protein import proceeds through the nuclear pore complex (NPC). Importin-beta mediates translocation via direct interaction with NPC components and carries importin-alpha with the NLS substrate from the cytoplasm into the nucleus. The import reaction is terminated by the direct binding of nuclear RanGTP to importin-beta which dissociates the importin heterodimer. Here, we analyse the sites of interaction on importin-beta for its multiple partners. Ran and importin-alpha respectively require residues 1-364 and 331-876 of importin-beta for binding. Thus, RanGTP-mediated release of importin-alpha from importin-beta is likely to be an active displacement rather than due to simple competition between Ran and importin-alpha for a common binding site. Importin-beta has at least two non-overlapping sites of interaction with the NPC, which could potentially be used sequentially during translocation. Our data also suggest that termination of import involves a transient release of importin-beta from the NPC. Importin-beta fragments which bind to the NPC, but not to Ran, resist this release mechanism. As would be predicted from this, these importin-beta mutants are very efficient inhibitors of NLS-dependent protein import. Surprisingly, however, they also inhibit M9 signal-mediated nuclear import as well as nuclear export of mRNA, U snRNA, and the NES-containing Rev protein. This suggests that mediators of these various transport events share binding sites on the NPC and/or that mechanisms exist to coordinate translocation through the NPC via different nucleocytoplasmic transport pathways.     

ATM activation and its recruitment to damaged DNA require binding to the C terminus of Nbs1

ATM has a central role in controlling the cellular responses to DNA damage. It and other phosphoinositide 3-kinase-related kinases (PIKKs) have giant helical HEAT repeat domains in their amino-terminal regions. The functions of these domains in PIKKs are not well understood. ATM activation in response to DNA damage appears to be regulated by the Mre11-Rad50-Nbs1 (MRN) complex, although the exact functional relationship between the MRN complex and ATM is uncertain. Here we show that two pairs of HEAT repeats in fission yeast ATM (Tel1) interact with an FXF/Y motif at the C terminus of Nbs1. This interaction resembles nucleoporin FXFG motif binding to HEAT repeats in importin-beta. Budding yeast Nbs1 (Xrs2) appears to have two FXF/Y motifs that interact with Tel1 (ATM). In Xenopus egg extracts, the C terminus of Nbs1 recruits ATM to damaged DNA, where it is subsequently autophosphorylated. This interaction is essential for ATM activation. A C-terminal 147-amino-acid fragment of Nbs1 that has the Mre11- and ATM-binding domains can restore ATM activation in an Nbs1-depleted extract. We conclude that an interaction between specific HEAT repeats in ATM and the C-terminal FXF/Y domain of Nbs1 is essential for ATM activation. We propose that conformational changes in the MRN complex that occur upon binding to damaged DNA are transmitted through the FXF/Y-HEAT interface to activate ATM. This interaction also retains active ATM at sites of DNA damage.     

The structure of the nuclear export receptor Cse1 in its cytosolic state reveals a closed conformation incompatible with cargo binding

Cse1 mediates nuclear export of importin alpha, the nuclear localization signal (NLS) import adaptor. We report the 3.1 A resolution structure of cargo-free Cse1, representing this HEAT repeat protein in its cytosolic state. Cse1 is compact, consisting of N- and C-terminal arches that interact to form a ring. Comparison with the structure of cargo-bound Cse1 shows a major conformational change leading to opening of the structure upon cargo binding. The largest structural changes occur within a hinge region centered at HEAT repeat 8. This repeat contains a conserved insertion that connects the RanGTP and importin alpha contact sites and that is essential for binding. In the cargo-free state, the RanGTP binding sites are occluded and the importin alpha sites are distorted. Mutations that destabilize the N- to C-terminal interaction uncouple importin alpha and Ran binding, suggesting that the closed conformation prevents association with importin alpha.     

P446L-importin-beta inhibits nuclear envelope assembly by sequestering nuclear envelope assembly factors to the microtubules

The P446L mutant Drosophila importin-beta (P446L-imp-beta) has been reported to prohibit--in dominant negative fashion--nuclear envelope (NE) assembly. Along elucidating the mode of action of P446L-imp-beta we studied in vitro NE assembly on Sepharose beads. While Drosophila embryo extracts support NE assembly over Sepharose beads coated with Ran, NE assembly does not take place in extracts supplied with exogenous P446L-imp-beta. A NE also forms over importin-beta-coated beads. Surprisingly, when immobilized to Sepharose beads P446L-imp-beta as efficiently recruits NE vesicles as normal importin-beta. The discrepancy in behavior of cytoplasmic and bead-bound P446L-imp-beta appears to be related to icreased--as compared to normal importin-beta--microtubule (MT) binding ability of P446L-imp-beta. While wild-type importin-beta is able to bind MTs and the binding decreases upon RanGTP interaction, P446L-imp-beta cannot be removed from the MTs by RanGTP. P446L-imp-beta, like normal importin-beta, binds some types of the nucleoporins that have been known to be required for NE assembly at the end of mitosis. It appears that the inhibitory effect of P446L-imp-beta on NE assembly is caused by sequestering some of the nucleoporins required for NE assembly to the MTs.     

The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus.

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran''s nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.     

Localized regulation of axonal RanGTPase controls retrograde injury signaling in peripheral nerve

Peripheral sensory neurons respond to axon injury by activating an importin-dependent retrograde signaling mechanism. How is this mechanism regulated? Here, we show that Ran GTPase and its associated effectors RanBP1 and RanGAP regulate the formation of importin signaling complexes in injured axons. A gradient of nuclear RanGTP versus cytoplasmic RanGDP is thought to be fundamental for the organization of eukaryotic cells. Surprisingly, we find RanGTP in sciatic nerve axoplasm, distant from neuronal cell bodies and nuclei, and in association with dynein and importin-alpha. Following injury, localized translation of RanBP1 stimulates RanGTP dissociation from importins and subsequent hydrolysis, thereby allowing binding of newly synthesized importin-beta to importin-alpha and dynein. Perturbation of RanGTP hydrolysis or RanBP1 blockade at axonal injury sites reduces the neuronal conditioning lesion response. Thus, neurons employ localized mechanisms of Ran regulation to control retrograde injury signaling in peripheral nerve.     

Binding dynamics of isolated nucleoporin repeat regions to importin-beta

The nuclear pore complex, through the interaction of its proteins with transport receptors, controls the transport of large molecules into and out of the cell's nucleus. There is ample evidence for proteins with FG sequence repeats playing an essential role in this control. Previous studies have elucidated binding spots for FG sequence repeats on the surface of the transport receptor importin-beta by X-ray crystallography and mutational studies. Molecular dynamics simulations have been performed to characterize the interaction of FG sequence repeats with the transport receptor. Observed binding spots have been verified and novel sites discovered, suggesting that importin-beta features many more binding spots than suspected so far. The observed binding spots are in accord with several models of nucleocytoplasmic transport, and the large number of binding spots on importin-beta may be necessary for the pore complex to distinguish between importin-beta and inert proteins, and to allow for its passage through the pore.     

Insights into the molecular mechanism of nuclear trafficking using nuclear transport factor 2 (NTF2)

Nuclear transport factor 2 (NTF2) mediates the nuclear import of RanGDP. The simplicity and specialization of this system, combined with the availability of crystal structures of NTF2, RanGDP and their complex, has facilitated the investigation of the molecular mechanism of its trafficking. NTF2 binds to both RanGDP and FxFG repeat-containing nucleoporins. Mutants engineered on the basis of structural information together with determination of binding constants have been used to dissect the roles of these interactions in transport. Thus, NTF2 binds to RanGDP sufficiently strongly for the complex to remain intact during transport through NPCs, but the interaction between NTF2 and FxFG nucleoporins is much more transient, which would enable NTF2 to move through the NPC by hopping from one repeat to another. An analogous nucleoporin hopping mechanism may also be used by carrier molecules of the importin-beta family to move through NPCs.     

Structural basis for the interaction between the Tap/NXF1 UBA domain and FG nucleoporins at 1A resolution

The mRNA nuclear export function of Tap/NXF1 requires interactions with nuclear pore proteins (nucleoporins) that contain characteristic Phe-Gly repeats based on FG, GLFG or FxFG cores separated by hydrophilic linkers. FG-nucleoporins bind the two most C-terminal domains of Tap, which have NTF2 and UBA folds, respectively. We used a combination of NMR and X-ray crystallography to define the interaction interface between Tap UBA and FxFG nucleoporins and show that it involves primarily the two aromatic rings of the FxFG core that bind in a hydrophobic surface depression centred on Tap Cys588. NMR evidence indicates that the same depression mediates the binding of GLFG nucleoporins, which we confirmed by demonstrating competition between the two classes of repeat for binding to Tap UBA. Moreover, modification of Cys588 reduced the binding of Tap UBA to both GLFG and FxFG nucleoporins as well as to nuclear envelopes. These data underscore the central role of the conserved FG-nucleoporin repeat cores in binding to Tap UBA and indicate that functional differences between different classes of nucleoporins depend more on their spatial distribution in nuclear pores than on their binding to different sites on Tap UBA.