Defective recognition of a nonclassical nuclear localization signal in neurodevelopmental disorders.

In this issue of Structure, Gonzalez et al. present the cryo-EM structure of Karyopherin-ß2 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.

Crystallographic data of an importin-α3 dimer in which the two protomers are bridged by a bipartite nuclear localization signal.

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, ß = 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).

Structures of importin-α bound to the wild-type and an internal deletion mutant of the bipartite nuclear localization signal of HIF-1α.

Hypoxia-inducible factor 1 (HIF-1) is a heterodimeric transcription factor that plays an important role as a master regulator of oxygen homeostasis. The activity of HIF-1 is regulated in part by dynamic intracellular trafficking of its α subunit (HIF-1α) that can shuttle between the nucleus and cytoplasm. It has been shown that nuclear localization of HIF-1α requires a variant of classic nuclear localization signal (NLS) and that an internal deletion of the amino acid residues (residues 724-751) in the NLS almost abolish the nuclear localization. Here we report the X-ray crystal structure of the nuclear import adaptor importin-α1 bound to the wild-type HIF-1α NLS at 1.8 Å resolution and of importin-α1 bound to the Δ724-751 mutant of the HIF-1α NLS at 1.9 Å resolution. In the wild-type structure, two basic clusters in the HIF-1α NLS made extensive interactions with importin-α1 on two sites (the major site and the minor site). In the mutant structure, the NLS residues still interacted extensively with the major site on importin-α1, but the interactions with the minor site were not observed. The structural data, together with computational analyses of binding free energies, indicate that the loss of the minor-site interactions inhibit nuclear accumulation of HIF-1α.

High-resolution structural analysis shows how different crystallographic environments can induce alternative modes of binding of a phosphotyrosine peptide to the SH2 domain of Fer tyrosine kinase.

Fes and Fes-related (Fer) protein tyrosine kinases (PTKs) comprise a subfamily of nonreceptor tyrosine kinases characterized by a unique multidomain structure composed of an N-terminal Fer/CIP4 homology-Bin/Amphiphysin/Rvs (F-BAR) domain, a central Src homology 2 (SH2) domain, and a C-terminal PTK domain. Fer is ubiquitously expressed, and upregulation of Fer has been implicated in various human cancers. The PTK activity of Fes has been shown to be positively regulated by the binding of phosphotyrosine-containing ligands to the SH2 domain. Here, the X-ray crystal structure of human Fer SH2 domain bound to a phosphopeptide that has D-E-pY-E-N-V-D sequence is reported at 1.37 å resolution. The asymmetric unit (ASU) contains six Fer-phosphopeptide complexes, and the structure reveals three distinct binding modes for the same phosphopeptide. At four out of the six binding sites in the ASU, the phosphopeptide binds to Fer SH2 domain in a type I ß-turn conformation, and this could be the optimal binding mode of this phosphopeptide. At the other two binding sites in the ASU, it appears that spatial proximity of neighboring SH2 domains in the crystal induces alternative modes of binding of this phosphopeptide.

Structural and biochemical characterization of the recognition of the 53BP1 nuclear localization signal by importin-α.

53BP1 (TP53-binding protein 1) plays a key role in DNA double-strand break repair by promoting non-homologous end joining (NHEJ) especially during G1 phase of the cell cycle. Nuclear import of 53BP1 is required for proper localization of 53BP1 and maintenance of genome integrity. 53BP1 has a classical bipartite nuclear localization signal (NLS) of sequence 1666-GKRKLITSEEERSPAKRGRKS-1686. Ser1678 within the 53BP1 NLS can be phosphorylated by CDK1/cyclin B, and a phosphomimetic substitution of Ser1678 with aspartate has been shown to negatively regulate nuclear import of 53BP1. Here, the X-ray crystal structures of the nuclear import adaptor importin-α1 bound to the wild-type 53BP1 NLS and the S1678D mutant of 53BP1 NLS are reported at resolutions of 1.9 and 1.7 Å, respectively. In the wild-type structure, not only the two basic clusters of the 53BP1 NLS but also the linker region between the basic clusters made extensive interactions with importin-α1. In the mutant structure, the linker region between the basic clusters in the 53BP1 NLS made fewer interactions with importin-α1 than those observed in the wild-type structure. However, biochemical binding assays using purified proteins showed that the 53BP1 mutation S1678D reduces the binding affinity to importin-α1 only to a modest extent. Implications of these findings for regulatory mechanism of 53BP1 nuclear import are discussed.

Crystal structure of the Xpo1p nuclear export complex bound to the SxFG/PxFG repeats of the nucleoporin Nup42p.

Xpo1p (yeast CRM1) is the major nuclear export receptor that carries a plethora of proteins and ribonucleoproteins from the nucleus to cytoplasm. The passage of the Xpo1p nuclear export complex through nuclear pore complexes (NPCs) is facilitated by interactions with nucleoporins (Nups) containing extensive repeats of phenylalanine-glycine (so-called FG repeats), although the precise role of each Nup in the nuclear export reaction remains incompletely understood. Here we report structural and biochemical characterization of the interactions between the Xpo1p nuclear export complex and the FG repeats of Nup42p, a nucleoporin localized at the cytoplasmic face of yeast NPCs and has characteristic SxFG/PxFG sequence repeat motif. The crystal structure of Xpo1p-PKI-Nup42p-Gsp1p-GTP complex identified three binding sites for the SxFG/PxFG repeats on HEAT repeats 14-20 of Xpo1p. Mutational analyses of Nup42p showed that the conserved serines and prolines in the SxFG/PxFG repeats contribute to Xpo1p-Nup42p binding. Our structural and biochemical data suggest that SxFG/PxFG-Nups such as Nup42p and Nup159p at the cytoplasmic face of NPCs provide high-affinity docking sites for the Xpo1p nuclear export complex in the terminal stage of NPC passage and that subsequent disassembly of the nuclear export complex facilitates recycling of free Xpo1p back to the nucleus.

Crystal structure of importin-α3 bound to the nuclear localization signal of Ran-binding protein 3.

Ran-binding protein 3 (RanBP3) is a primarily nuclear Ran-binding protein that functions as an accessory factor in the Ran GTPase system. RanBP3 associates with Ran-specific nucleotide exchange factor RCC1 and enhances its catalytic activity towards Ran. RanBP3 also promotes CRM1-mediated nuclear export as well as CRM1-independent nuclear export of ß-catenin, Smad2, and Smad3. Nuclear import of RanBP3 is dependent on the nuclear import adaptor protein importin-α and, RanBP3 is imported more efficiently by importin-α3 than by other members of the importin-α family. Protein kinase signaling pathways control nucleocytoplasmic transport through phosphorylation of RanBP3 at Ser58, immediately C-terminal to the nuclear localization signal (NLS) in the N-terminal region of RanBP3. Here we report the crystal structure of human importin-α3 bound to an N-terminal fragment of human RanBP3 containing the NLS sequence that is necessary and sufficient for nuclear import. The structure reveals that RanBP3 binds to importin-α3 residues that are strictly conserved in all seven isoforms of human importin-α at the major NLS-binding site, indicating that the region of importin-α outside the NLS-binding site, possibly the autoinhibotory importin-ß1-binding domain, may be the key determinant for the preferential binding of RanBP3 to importin-α3. Computational docking simulation indicates that phosphorylation of RanBP3 at Ser58 could potentially stabilize the association of RanBP3 with importin-α through interactions between the phosphate moiety of phospho-Ser58 of RanBP3 and a cluster of basic residues (Arg96 and Lys97 in importin-α3) on armadillo repeat 1 of importin-α.

Structure and dimerization of the catalytic domain of the protein phosphatase Cdc14p, a key regulator of mitotic exit in Saccharomyces cerevisiae.

In the budding yeast Saccharomyces cerevisiae, the protein phosphatase Cdc14p orchestrates various events essential for mitotic exit. We have determined the X-ray crystal structures at 1.85 Å resolution of the catalytic domain of Cdc14p in both the apo state, and as a complex with S160-phosphorylated Swi6p peptide. Each asymmetric unit contains two Cdc14p chains arranged in an intimately associated homodimer, consistent with its oligomeric state in solution. The dimerization interface is located on the backside of the substrate-binding cleft. Structure-based mutational analyses indicate that the dimerization of Cdc14p is required for normal growth of yeast cells.

Crystal structure of human WBSCR16, an RCC1-like protein in mitochondria.

WBSCR16 (Williams-Beuren Syndrome Chromosomal Region 16) gene is located in a large deletion region of Williams-Beuren syndrome (WBS), which is a neurodevelopmental disorder. Although the relationship between WBSCR16 and WBS remains unclear, it has been reported that WBSCR16 is a member of a functional module that regulates mitochondrial 16S rRNA abundance and intra-mitochondrial translation. WBSCR16 has RCC1 (Regulator of Chromosome Condensation 1)-like amino acid sequence repeats but the function of WBSCR16 appears to be different from that of other RCC1 superfamily members. Here, we demonstrate that WBSCR16 localizes to mitochondria in HeLa cells, and report the crystal structure of WBSCR16 determined to 2.0 Å resolution using multi-wavelength anomalous diffraction. WBSCR16 adopts the seven-bladed ß-propeller fold characteristic of RCC1-like proteins. A comparison of the WBSCR16 structure with that of RCC1 and other RCC1-like proteins reveals that, although many of the residues buried in the core of the ß-propeller are highly conserved, the surface residues are poorly conserved and conformationally divergent.

Crystal structure of importin-α bound to the nuclear localization signal of Epstein-Barr virus EBNA-LP protein.

Epstein-Barr virus EBNA-LP protein is a transcriptional coactivator of EBNA2. Efficient nuclear localization of EBNA-LP is essential for cooperation with EBNA2. Here, we report the crystal structure of the nuclear import adaptor importin-α1 bound to the nuclear localization signal (NLS) of EBNA-LP that shows EBNA-LP residues 44-RRVRRR-49 binding to the major NLS-binding site at the P0-P5 positions. In contrast to previously characterized classical NLSs that invariably have a basic residue [either lysine (in the vast majority of cases) or arginine] at the P2 position, the EBNA-LP NLS is unique in that it has valine at the P2 position. The loss of the critical P2 lysine (or arginine) is compensated by arginine at the P0 position in the EBNA-LP NLS.

Structural basis for the regulation of nuclear import of Epstein-Barr virus nuclear antigen 1 (EBNA1) by phosphorylation of the nuclear localization signal.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in every EBV-positive tumor and is essential for the maintenance, replication, and transcription of the EBV genome in the nucleus of host cells. EBNA1 is a serine phosphoprotein, and it has been shown that phosphorylation of S385 in the nuclear localization signal (NLS) of EBNA1 increases the binding affinity to the nuclear import adaptor importin-α1 as well as importin-α5, and stimulates nuclear import of EBNA1. To gain insights into how phosphorylation of the EBNA1 NLS regulates nuclear import, we have determined the crystal structures of two peptide complexes of importin-α1: one with S385-phosphorylated EBNA1 NLS peptide, determined at 2.0 Å resolution, and one with non-phosphorylated EBNA1 NLS peptide, determined at 2.2 Å resolution. The structures show that EBNA1 NLS binds to the major and minor NLS-binding sites of importin-α1, and indicate that the binding affinity of the EBNA1 NLS to the minor NLS-binding site could be enhanced by phosphorylation of S385 through electrostatic interaction between the phosphate group of phospho-S385 and K392 of importin-α1 (corresponding to R395 of importin-α5) on armadillo repeat 8.

Structures of the Karyopherins Kap121p and Kap60p Bound to the Nuclear Pore-Targeting Domain of the SUMO Protease Ulp1p.

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-ß-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.

Mechanistic Insights from Structural Analyses of Ran-GTPase-Driven Nuclear Export of Proteins and RNAs.

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-ß 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.

Structure of importin-α bound to a non-classical nuclear localization signal of the influenza A virus nucleoprotein.

A non-classical nuclear localization signal (ncNLS) of influenza A virus nucleoprotein (NP) is critical for nuclear import of viral genomic RNAs that transcribe and replicate in the nucleus of infected cells. Here we report a 2.3 Å resolution crystal structure of mouse importin-α1 in complex with NP ncNLS. The structure reveals that NP ncNLS binds specifically and exclusively to the minor NLS-binding site of importin-α. Structural and functional analyses identify key binding pockets on importin-α as potential targets for antiviral drug development. Unlike many other NLSs, NP ncNLS binds to the NLS-binding domain of importin-α weakly with micromolar affinity. These results suggest that a modest inhibitor with low affinity to importin-α could have anti-influenza activity with minimal cytotoxicity.

Crystal structure of the karyopherin Kap121p bound to the extreme C-terminus of the protein phosphatase Cdc14p.

In Saccharomyces cerevisiae, the protein phosphatase Cdc14p is an antagonist of mitotic cyclin-dependent kinases and is a key regulator of late mitotic events such as chromosome segregation, spindle disassembly and cytokinesis. The activity of Cdc14p is controlled by cell-cycle dependent changes in its association with its competitive inhibitor Net1p (also known as Cfi1p) in the nucleolus. For most of the cell cycle up to metaphase, Cdc14p is sequestered in the nucleolus in an inactive state. During anaphase, Cdc14p is released from Net1p, spreads into the nucleus and cytoplasm, and dephosphorylates key mitotic targets. Although regulated nucleocytoplasmic shuttling of Cdc14p has been suggested to be important for exit from mitosis, the mechanism underlying Cdc14p nuclear trafficking remains poorly understood. Here we show that the C-terminal region (residues 517-551) of Cdc14p can function as a nuclear localization signal (NLS) in vivo and also binds to Kap121p (also known as Pse1p), an essential nuclear import carrier in yeast, in a Gsp1p-GTP-dependent manner in vitro. Moreover we report a crystal structure, at 2.4 Å resolution, of Kap121p bound to the C-terminal region of Cdc14p. The structure and structure-based mutational analyses suggest that either the last five residues at the extreme C-terminus of Cdc14p (residues 547-551; Gly-Ser-Ile-Lys-Lys) or adjacent residues with similar sequence (residues 540-544; Gly-Gly-Ile-Arg-Lys) can bind to the NLS-binding site of Kap121p, with two residues (Ile in the middle and Lys at the end of the five residues) of Cdc14p making key contributions to the binding specificity. Based on comparison with other structures of Kap121p-ligand complexes, we propose "IK-NLS" as an appropriate term to refer to the Kap121p-specific NLS.

Structural insights into how Yrb2p accelerates the assembly of the Xpo1p nuclear export complex.

Proteins and ribonucleoproteins containing a nuclear export signal (NES) assemble with the exportin Xpo1p (yeast CRM1) and Gsp1p-GTP (yeast Ran-GTP) in the nucleus and exit through the nuclear pore complex. In the cytoplasm, Yrb1p (yeast RanBP1) displaces NES from Xpo1p. Efficient export of NES-cargoes requires Yrb2p (yeast RanBP3), a primarily nuclear protein containing nucleoporin-like phenylalanine-glycine (FG) repeats and a low-affinity Gsp1p-binding domain (RanBD). Here, we show that Yrb2p strikingly accelerates the association of Gsp1p-GTP and NES to Xpo1p. We have solved the crystal structure of the Xpo1p-Yrb2p-Gsp1p-GTP complex, a key assembly intermediate that can bind cargo rapidly. Although the NES-binding cleft of Xpo1p is closed in this intermediate, our data suggest that preloading of Gsp1p-GTP onto Xpo1p by Yrb2p, conformational flexibility of Xpo1p, and the low affinity of RanBD enable active displacement of Yrb2p RanBD by NES to occur effectively. The structure also reveals the major binding sites for FG repeats on Xpo1p.

Structural basis for cell-cycle-dependent nuclear import mediated by the karyopherin Kap121p.

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.

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.

Structures of the pleckstrin homology domain of Saccharomyces cerevisiae Avo1 and its human orthologue Sin1, an essential subunit of TOR complex 2.

In eukaryotes, multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2) function as major regulators of cell growth, metabolism and ageing. The C-terminal domain of the Saccharomyces cerevisiae TORC2 component Avo1 is required for plasma-membrane localization of TORC2 and is essential for yeast viability. X-ray crystal structures of the C-terminal domain of Avo1 and of its human orthologue Sin1 have been determined. The structures show that the C-termini of Avo1 and Sin1 both have the pleckstrin homology (PH) domain fold. Comparison with known PH-domain structures suggests a putative binding site for phosphoinositides.