53BP1 (TP53-binding protein 1), a key player in DNA double-strand break repair, has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686 that binds to ...importin-α, a nuclear import adaptor protein. Nucleoporin Nup153 is involved in nuclear import of 53BP1, and the binding of Nup153 to importin-α has been proposed to promote efficient import of classical NLS-containing proteins. Here, the ARM-repeat domain of human importin-α3 bound to 53BP1 NLS was crystallized in the presence of a synthetic peptide corresponding to the extreme C-terminus of Nup153 (sequence: 1459-GTSFSGRKIKTAVRRRK-1475). The crystal belonged to space group I2, with unit-cell parameters a = 95.70, b = 79.60, c = 117.44 Å, β = 95.57°. The crystal diffracted X-rays to 1.9 Å resolution, and the structure was solved by molecular replacement. The asymmetric unit contained two molecules of importin-α3 and two molecules of 53BP1 NLS. Although no convincing density was observed for the Nup153 peptide, the electron density corresponding to 53BP1 NLS was unambiguous and continuous along the entire length of the bipartite NLS. The structure revealed a novel dimer of importin-α3, in which two protomers of importin-α3 are bridged by the bipartite NLS of 53BP1. In this structure, the upstream basic cluster of the NLS is bound to the minor NLS-binding site of one protomer of importin-α3, whereas the downstream basic cluster of the same chain of NLS is bound to the major NLS-binding site of another protomer of importin-α3. This quaternary structure is distinctly different from the previously determined crystal structure of mouse importin-α1 bound to the 53BP1 NLS. The atomic coordinates and structure factors have been deposited in the Protein Data Bank (accession code 8HKW).
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.
Kap121p (also known as Pse1p) is an essential karyopherin that mediates nuclear import of a plethora of cargoes including cell cycle regulators, transcription factors, and ribosomal proteins in ...Saccharomyces cerevisiae. It has been proposed that the spindle assembly checkpoint signaling triggers molecular rearrangements of nuclear pore complexes and thereby arrests Kap121p-mediated nuclear import at metaphase, while leaving import mediated by other karyopherins unaffected. The Kap121p-specific import inhibition is required for normal progression through mitosis. To understand the structural basis for Kap121p-mediated nuclear import and its unique regulatory mechanism during mitosis, we determined crystal structures of Kap121p in isolation and also in complex with either its import cargoes or nucleoporin Nup53p or RanGTP. Kap121p has a superhelical structure composed of 24 HEAT repeats. The structures of Kap121p–cargo complexes define a non-conventional nuclear localization signal (NLS) that has a consensus sequence of KV/IxKx1-2K/H/R. The structure of Kap121p–Nup53p complex shows that cargo and Nup53p compete for the same high-affinity binding site, explaining how Nup53p binding forces cargo release when the Kap121p-binding site of Nup53p is exposed during mitosis. Comparison of the NLS and RanGTP complexes reveals that RanGTP binding not only occludes the cargo-binding site but also forces Kap121p into a conformation that is incompatible with NLS recognition.
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► The inhibition of Kap121p-mediated nuclear import during mitosis is required for normal progression of cell cycle. ► The structures of Kap121p–cargo complexes define a novel NLS recognition mechanism. ► The structure of Kap121p–Nup53p complex explains how Nup53p inhibits import during mitosis. ► The structure of Kap121p–RanGTP complex shows how Ran terminates nuclear import. ► The structures advance understanding of how various nuclear import pathways are differentially regulated.
In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-β2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ...ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.
In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-β2 bound to the proline-tyrosine nuclear localization signal (PY-NLS) of heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2). The structure advances our understanding of not only the diversity of PY-NLSs but also the pathogenic mechanisms arising from HNRNPH2 variants.
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.
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► Mechanism of cooperative RanGTP and cargo binding to CRM1 has remained obscure. ► We report a 2.1-Å-resolution crystal structure of unliganded S. cerevisiae CRM1. ► The HEAT9 loop is primarily responsible for autoinhibition of CRM1. ► The C-terminus of CRM1 reinforces autoinhibition by the HEAT9 loop. ► The structure advances understanding of allosteric coupling in CRM1.
Understanding how macromolecules are rapidly exchanged between the nucleus and the cytoplasm through nuclear pore complexes is a fundamental problem in biology. Exportins are Ran-GTPase-dependent ...nuclear transport factors that belong to the karyopherin-β family and mediate nuclear export of a plethora of proteins and RNAs, except for bulk mRNA nuclear export. Exportins bind cargo macromolecules in a Ran-GTP-dependent manner in the nucleus, forming exportin-cargo-Ran-GTP complexes (nuclear export complexes). Transient weak interactions between exportins and nucleoporins containing characteristic FG (phenylalanine-glycine) repeat motifs facilitate nuclear pore complex passage of nuclear export complexes. In the cytoplasm, nuclear export complexes are disassembled, thereby releasing the cargo. GTP hydrolysis by Ran promoted in the cytoplasm makes the disassembly reaction virtually irreversible and provides thermodynamic driving force for the overall export reaction. In the past decade, X-ray crystallography of some of the exportins in various functional states coupled with functional analyses, single-particle electron microscopy, molecular dynamics simulations, and small-angle solution X-ray scattering has provided rich insights into the mechanism of cargo binding and release and also begins to elucidate how exportins interact with the FG repeat motifs. The knowledge gained from structural analyses of nuclear export is being translated into development of clinically useful inhibitors of nuclear export to treat human diseases such as cancer and influenza.
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•Exportins are Ran-GTPase-dependent nuclear export factors for proteins and RNAs.•Structures of exportins in various functional states have been determined.•Conformational changes in exportins couple cargo binding to Ran-GTP binding.•RanBPs can accelerate assembly/disassembly of nuclear export complexes.•Inhibitors of nuclear export are being developed and may be clinically useful.
The nuclear import and export of macromolecular cargoes through nuclear pore complexes is mediated primarily by carriers such as importin-beta. Importins carry cargoes into the nucleus, whereas ...exportins carry cargoes to the cytoplasm. Transport is orchestrated by nuclear RanGTP, which dissociates cargoes from importins, but conversely is required for cargo binding to exportins. Here we present the 2.0 A crystal structure of the nuclear export complex formed by exportin Cse1p complexed with its cargo (Kap60p) and RanGTP, thereby providing a structural framework for understanding nuclear protein export and the different functions of RanGTP in export and import. In the complex, Cse1p coils around both RanGTP and Kap60p, stabilizing the RanGTP-state and clamping the Kap60p importin-beta-binding domain, ensuring that only cargo-free Kap60p is exported. Mutagenesis indicated that conformational changes in exportins couple cargo binding to high affinity for RanGTP, generating a spring-loaded molecule to facilitate disassembly of the export complex following GTP hydrolysis in the cytoplasm.
The budding yeast small ubiquitin-like modifier (SUMO) protease Ulp1p catalyzes both the processing of newly synthesized SUMO to its mature form and the deconjugation of SUMO from target proteins, ...thereby regulating a wide range of cellular processes including cell division, DNA repair, DNA replication, transcription, and mRNA quality control. Ulp1p is localized primarily at the nuclear pore complex (NPC) through interactions involving the karyopherins Kap121p and Kap95p–Kap60p heterodimer and a subset of nuclear pore-associated proteins. The sequestration of Ulp1p at the nuclear periphery is crucial for the proper control of protein desumoylation. To gain insights into the role of the karyopherins in regulating the localization of Ulp1p, we have determined the crystal structures of Kap121p and Kap60p bound to the N-terminal non-catalytic domain of Ulp1p that is necessary and sufficient for NPC targeting. Contrary to a previous proposal that Ulp1p is tethered to the transport channel of the NPC through unconventional interactions with the karyopherins, our structures reveal that Ulp1p has canonical nuclear localization signals (NLSs): (1) an isoleucine-lysine-NLS (residues 51–55) that binds to the NLS-binding site of Kap121p, and (2) a classical bipartite NLS (residues 154–172) that binds to the major and minor NLS-binding sites of Kap60p. Ulp1p also binds Kap95p directly, and the Ulp1p–Kap95p binding is enhanced by the importin-β-binding domain of Kap60p. GTP-bound Gsp1p (the yeast Ran ortholog) and the exportin Cse1p cooperate to release Ulp1p from the karyopherins, indicating that the stable sequestration of Ulp1p to the NPC would require a karyopherin-independent mechanism to anchor Ulp1p at the NPC.
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•The NPC-associated SUMO protease Ulp1p regulates numerous cellular processes.•The karyopherins bind the non-catalytic domain of Ulp1p and target Ulp1p to the NPC.•Structures of the Kap121p–Ulp1p complex and the Kap60p–Ulp1p complex are reported.•The non-catalytic domain of Ulp1p contains canonical NLSs.•GTP-bound Gsp1p and Cse1p cooperate to release Ulp1p from the karyopherins.
► MAL has a bipartite NLS that binds to Impα in an extended conformation. ► Mutational analyses verified the functional significance of MAL–Impα interactions. ► Induced folding and NLS-masking by ...G-actins inhibit nuclear import of MAL.
The coordination of cytoskeletal actin dynamics with gene expression reprogramming is emerging as a crucial mechanism to control diverse cellular processes, including cell migration, differentiation and neuronal circuit assembly. The actin-binding transcriptional coactivator MAL (also known as MRTF-A/MKL1/BSAC) senses G-actin concentration and transduces Rho GTPase signals to serum response factor (SRF). MAL rapidly shuttles between the cytoplasm and the nucleus in unstimulated cells but Rho-induced depletion of G-actin leads to MAL nuclear accumulation and activation of transcription of SRF:MAL-target genes. Although the molecular and structural basis of actin-regulated nucleocytoplasmic shuttling of MAL is not understood fully, it is proposed that nuclear import of MAL is mediated by importin α/β heterodimer, and that G-actin competes with importin α/β for the binding to MAL. Here we present structural, biochemical and cell biological evidence that MAL has a classical bipartite nuclear localization signal (NLS) in the N-terminal ‘RPEL’ domain containing Arg-Pro-X-X-X-Glu-Leu (RPEL) motifs. The NLS residues of MAL adopt an extended conformation and bind along the surface groove of importin-α, interacting with the major- and minor-NLS binding sites. We also present a crystal structure of wild-type MAL RPEL domain in complex with five G-actins. Comparison of the importin-α- and actin-complexes revealed that the binding of G-actins to MAL is associated with folding of NLS residues into a helical conformation that is inappropriate for importin-α recognition.
Nuclear import of proteins containing classical nuclear localization signals (NLS) is mediated by the importin‐α:β complex that binds cargo in the cytoplasm and facilitates its passage through ...nuclear pores, after which nuclear RanGTP dissociates the import complex and the importins are recycled. In vertebrates, import is stimulated by nucleoporin Nup50, which has been proposed to accompany the import complex through nuclear pores. However, we show here that the Nup50 N‐terminal domain actively displaces NLSs from importin‐α, which would be more consistent with Nup50 functioning to coordinate import complex disassembly and importin recycling. The crystal structure of the importin‐α:Nup50 complex shows that Nup50 binds at two sites on importin‐α. One site overlaps the secondary NLS‐binding site, whereas the second extends along the importin‐α C‐terminus. Mutagenesis indicates that interaction at both sites is required for Nup50 to displace NLSs. The Cse1p:Kap60p:RanGTP complex structure suggests how Nup50 is then displaced on formation of the importin‐α export complex. These results provide a rationale for understanding the series of interactions that orchestrate the terminal steps of nuclear protein import.
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