RESUMO
INTRODUCTION The subcellular compartmentalization of eukaryotic cells requires selective transport of folded proteins and protein-nucleic acid complexes. Embedded in nuclear envelope pores, which are generated by the circumscribed fusion of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) are the sole bidirectional gateways for nucleocytoplasmic transport. The ~110-MDa human NPC is an ~1000-protein assembly that comprises multiple copies of ~34 different proteins, collectively termed nucleoporins. The symmetric core of the NPC is composed of an inner ring encircling the central transport channel and outer rings formed by Yshaped coat nucleoporin complexes (CNCs) anchored atop both sides of the nuclear envelope. The outer rings are decorated with compartmentspecific asymmetric nuclear basket and cytoplasmic filament nucleoporins, which establish transport directionality and provide docking sites for transport factors and the small guanosine triphosphatase Ran. The cytoplasmic filament nucleoporins also play an essential role in the irreversible remodeling of messenger ribonucleoprotein particles (mRNPs) as they exit the central transport channel. Unsurprisingly, the NPC's cytoplasmic face represents a hotspot for diseaseassociated mutations and is commonly targeted by viral virulence factors. RATIONALE Previous studies established a near-atomic composite structure of the human NPC's symmetric core by combining (i) biochemical reconstitution to elucidate the interaction network between symmetric nucleoporins, (ii) crystal and single-particle cryo-electron microscopy structure determination of nucleoporins and nucleoporin complexes to reveal their three-dimensional shape and the molecular details of their interactions, (iii) quantitative docking in cryo-electron tomography (cryo-ET) maps of the intact human NPC to uncover nucleoporin stoichiometry and positioning, and (iv) cellbased assays to validate the physiological relevance of the biochemical and structural findings. In this work, we extended our approach to the cytoplasmic filament nucleoporins to reveal the near-atomic architecture of the cytoplasmic face of the human NPC. RESULTS Using biochemical reconstitution, we elucidated the protein-protein and protein-RNA interaction networks of the human and Chaetomium thermophilum cytoplasmic filament nucleoporins, establishing an evolutionarily conserved heterohexameric cytoplasmic filament nucleoporin complex (CFNC) held together by a central heterotrimeric coiledcoil hub that tethers two separate mRNPremodeling complexes. Further biochemical analysis and determination of a series of crystal structures revealed that the metazoanspecific cytoplasmic filament nucleoporin NUP358 is composed of 16 distinct domains, including an Nterminal Sshaped αhelical solenoid followed by a coiledcoil oligomerization element, numerous Raninteracting domains, an E3 ligase domain, and a Cterminal prolylisomerase domain. Physiologically validated quantitative docking into cryo-ET maps of the intact human NPC revealed that pentameric NUP358 bundles, conjoined by the oligomerization element, are anchored through their Nterminal domains to the central stalk regions of the CNC, projecting flexibly attached domains as far as ~600 Å into the cytoplasm. Using cellbased assays, we demonstrated that NUP358 is dispensable for the architectural integrity of the assembled interphase NPC and RNA export but is required for efficient translation. After NUP358 assignment, the remaining 4-shaped cryoET density matched the dimensions of the CFNC coiledcoil hub, in close proximity to an outer-ring NUP93. Whereas the N-terminal NUP93 assembly sensor motif anchors the properly assembled related coiledcoil channel nucleoporin heterotrimer to the inner ring, biochemical reconstitution confirmed that the NUP93 assembly sensor is reused in anchoring the CFNC to the cytoplasmic face of the human NPC. By contrast, two C. thermophilum CFNCs are anchored by a divergent mechanism that involves assembly sensors located in unstructured portions of two CNC nucleoporins. Whereas unassigned cryoET density occupies the NUP358 and CFNC binding sites on the nuclear face, docking of the nuclear basket component ELYS established that the equivalent position on the cytoplasmic face is unoccupied, suggesting that mechanisms other than steric competition promote asymmetric distribution of nucleoporins. CONCLUSION We have substantially advanced the biochemical and structural characterization of the asymmetric nucleoporins' architecture and attachment at the cytoplasmic and nuclear faces of the NPC. Our nearatomic composite structure of the human NPC's cytoplasmic face provides a biochemical and structural framework for elucidating the molecular basis of mRNP remodeling, viral virulence factor interference with NPC function, and the underlying mechanisms of nucleoporin diseases at the cytoplasmic face of the NPC. [Figure: see text].
Assuntos
Citoplasma , Proteínas Fúngicas , Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Transporte de RNA , RNA Mensageiro , Chaetomium , Microscopia Crioeletrônica , Citoplasma/química , Proteínas Fúngicas/química , Humanos , Chaperonas Moleculares/química , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , RNA Mensageiro/metabolismoRESUMO
MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread posttranscriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. We found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings support a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin-proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals.
Assuntos
Proteínas Argonautas/metabolismo , MicroRNAs/metabolismo , Estabilidade de RNA , RNA Longo não Codificante/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elonguina/genética , Elonguina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Células K562 , Camundongos , Células NIH 3T3 , Proteólise , RNA Longo não Codificante/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
The nuclear pore complex (NPC) serves as the sole bidirectional gateway of macromolecules in and out of the nucleus. Owing to its size and complexity (â¼1,000 protein subunits, â¼110 MDa in humans), the NPC has remained one of the foremost challenges for structure determination. Structural studies have now provided atomic-resolution crystal structures of most nucleoporins. The acquisition of these structures, combined with biochemical reconstitution experiments, cross-linking mass spectrometry, and cryo-electron tomography, has facilitated the determination of the near-atomic overall architecture of the symmetric core of the human, fungal, and algal NPCs. Here, we discuss the insights gained from these new advances and outstanding issues regarding NPC structure and function. The powerful combination of bottom-up and top-down approaches toward determining the structure of the NPC offers a paradigm for uncovering the architectures of other complex biological machines to near-atomic resolution.
Assuntos
Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Eucariotos/metabolismo , Eucariotos/ultraestrutura , Humanos , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Subunidades Proteicas , RNA Mensageiro/metabolismoRESUMO
The nuclear pore complex (NPC) controls the passage of macromolecules between the nucleus and cytoplasm, but how the NPC directly participates in macromolecular transport remains poorly understood. In the final step of mRNA export, the DEAD-box helicase DDX19 is activated by the nucleoporins Gle1, Nup214, and Nup42 to remove Nxf1â¢Nxt1 from mRNAs. Here, we report crystal structures of Gle1â¢Nup42 from three organisms that reveal an evolutionarily conserved binding mode. Biochemical reconstitution of the DDX19 ATPase cycle establishes that human DDX19 activation does not require IP6, unlike its fungal homologs, and that Gle1 stability affects DDX19 activation. Mutations linked to motor neuron diseases cause decreased Gle1 thermostability, implicating nucleoporin misfolding as a disease determinant. Crystal structures of human Gle1â¢Nup42â¢DDX19 reveal the structural rearrangements in DDX19 from an auto-inhibited to an RNA-binding competent state. Together, our results provide the foundation for further mechanistic analyses of mRNA export in humans.
Assuntos
Poro Nuclear/química , Poro Nuclear/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular , Chaetomium/genética , Chaetomium/metabolismo , RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Poro Nuclear/genética , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ácido Fítico/metabolismo , Transporte de RNA , RNA Mensageiro/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The nuclear pore complex (NPC) controls the transport of macromolecules between the nucleus and cytoplasm, but its molecular architecture has thus far remained poorly defined. We biochemically reconstituted NPC core protomers and elucidated the underlying protein-protein interaction network. Flexible linker sequences, rather than interactions between the structured core scaffold nucleoporins, mediate the assembly of the inner ring complex and its attachment to the NPC coat. X-ray crystallographic analysis of these scaffold nucleoporins revealed the molecular details of their interactions with the flexible linker sequences and enabled construction of full-length atomic structures. By docking these structures into the cryoelectron tomographic reconstruction of the intact human NPC and validating their placement with our nucleoporin interactome, we built a composite structure of the NPC symmetric core that contains ~320,000 residues and accounts for ~56 megadaltons of the NPC's structured mass. Our approach provides a paradigm for the structure determination of similarly complex macromolecular assemblies.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Mapas de Interação de Proteínas , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Microscopia Crioeletrônica , Cristalografia por Raios X , Citoplasma/metabolismo , Tomografia com Microscopia Eletrônica , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Dados de Sequência Molecular , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. We present the reconstitution and interdisciplinary analyses of the ~425-kilodalton inner ring complex (IRC), which forms the central transport channel and diffusion barrier of the NPC, revealing its interaction network and equimolar stoichiometry. The Nsp1â¢Nup49â¢Nup57 channel nucleoporin heterotrimer (CNT) attaches to the IRC solely through the adaptor nucleoporin Nic96. The CNTâ¢Nic96 structure reveals that Nic96 functions as an assembly sensor that recognizes the three-dimensional architecture of the CNT, thereby mediating the incorporation of a defined CNT state into the NPC. We propose that the IRC adopts a relatively rigid scaffold that recruits the CNT to primarily form the diffusion barrier of the NPC, rather than enabling channel dilation.
Assuntos
Chaetomium/ultraestrutura , Proteínas Fúngicas/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares/ultraestrutura , Poro Nuclear/ultraestrutura , Proteínas Nucleares/ultraestrutura , Sequência de Aminoácidos , Chaetomium/metabolismo , Proteínas Fúngicas/química , Dados de Sequência Molecular , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Proteínas Nucleares/química , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
The nuclear pore complex (NPC) constitutes the sole gateway for bidirectional nucleocytoplasmic transport. Despite half a century of structural characterization, the architecture of the NPC remains unknown. Here we present the crystal structure of a reconstituted ~400-kilodalton coat nucleoporin complex (CNC) from Saccharomyces cerevisiae at a 7.4 angstrom resolution. The crystal structure revealed a curved Y-shaped architecture and the molecular details of the coat nucleoporin interactions forming the central "triskelion" of the Y. A structural comparison of the yeast CNC with an electron microscopy reconstruction of its human counterpart suggested the evolutionary conservation of the elucidated architecture. Moreover, 32 copies of the CNC crystal structure docked readily into a cryoelectron tomographic reconstruction of the fully assembled human NPC, thereby accounting for ~16 megadalton of its mass.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestrutura , Cristalografia por Raios X , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Nucleocytoplasmic transport is facilitated by nuclear pore complexes (NPCs), which are massive proteinaceous transport channels embedded in the nuclear envelope. Nup192 is a major component of an adaptor nucleoporin subcomplex proposed to link the NPC coat with the central transport channel. Here, we present the structure of the â¼110-kDa N-terminal domain (NTD) of Nup192 at 2.7-Å resolution. The structure reveals an open ring-shaped architecture composed of Huntingtin, EF3, PP2A, and TOR1 (HEAT) and Armadillo (ARM) repeats. A comparison of different conformations indicates that the NTD consists of two rigid halves connected by a flexible hinge. Unexpectedly, the two halves of the ring are structurally related to karyopherin-α (Kap-α) and ß-karyopherin family members. Biochemically, we identify a conserved patch that binds an unstructured segment in Nup53 and show that a C-terminal tail region binds to a putative helical fragment in Nic96. The Nup53 segment that binds Nup192 is a classical nuclear localization-like sequence that interacts with Kap-α in a mutually exclusive and mechanistically distinct manner. The disruption of the Nup53 and Nic96 binding sites in vivo yields growth and mRNA export defects, revealing their critical role in proper NPC function. Surprisingly, both interactions are dispensable for NPC localization, suggesting that Nup192 possesses another nucleoporin interaction partner. These data indicate that the structured domains in the adaptor nucleoporin complex are held together by peptide interactions that resemble those found in karyopherinâ¢cargo complexes and support the proposal that the adaptor nucleoporins arose from ancestral karyopherins.
Assuntos
Evolução Molecular , Carioferinas/genética , Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Calorimetria , Cristalografia por Raios X , Hibridização in Situ Fluorescente , Carioferinas/química , Mutagênese Sítio-Dirigida , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Plasmodium falciparum erythrocyte invasion is dependent on high affinity recognition of sialic acid on cell surface receptors. The erythrocyte binding-like (EBL) family of invasion ligands mediates recognition of sialic acid on erythrocyte glycoproteins. Erythrocyte-binding antigen-140 (PfEBA-140/BAEBL) is a critical EBL ligand that binds sialic acid on its receptor glycophorin C. We present here the crystal structure of the two-domain receptor-binding region of PfEBA-140 in complex with a glycan containing sialic acid. The structure identifies two glycan-binding pockets unique to PfEBA-140 and not shared by other EBL ligands. Specific molecular interactions that enable receptor engagement are identified and reveal that the glycan binding mode is distinct from that of apicomplexan and viral cell surface recognition ligands as well as host immune factors that bind sialic acid. Erythrocyte binding experiments elucidated essential glycan contact residues and identified divergent functional roles for each receptor-binding site. One of four polymorphisms proposed to affect receptor binding was localized to a glycan-binding site, providing a structural basis for altered erythrocyte engagement. The studies described here provide the first full description of sialic acid-dependent molecular interactions at the P. falciparum erythrocyte invasion interface and define a framework for development of PfEBA-140-based therapeutics, vaccines, and diagnostics assessing vaccine efficacy and natural immunity to infection.
Assuntos
Proteínas de Transporte/metabolismo , Eritrócitos/metabolismo , Glicoforinas/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Sítios de Ligação , Proteínas de Transporte/genética , Eritrócitos/parasitologia , Glicoforinas/genética , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/uso terapêutico , Malária Falciparum/genética , Malária Falciparum/metabolismo , Malária Falciparum/prevenção & controle , Proteínas de Membrana , Ácido N-Acetilneuramínico/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genéticaRESUMO
The nuclear pore complex is the sole mediator of bidirectional transport between the nucleus and cytoplasm. Nup358 is a metazoan-specific nucleoporin that localizes to the cytoplasmic filaments and provides several binding sites for the mobile nucleocytoplasmic transport machinery. Here we present the crystal structure of the C-terminal domain (CTD) of Nup358 at 1.75Å resolution. The structure reveals that the CTD adopts a cyclophilin-like fold with a non-canonical active-site configuration. We determined biochemically that the CTD possesses weak peptidyl-prolyl isomerase activity and show that the active-site cavity mediates a weak association with the human immunodeficiency virus-1 capsid protein, supporting its role in viral infection. Overall, the surface is evolutionarily conserved, suggesting that the CTD serves as a protein-protein interaction platform. However, we demonstrate that the CTD is dispensable for nuclear envelope localization of Nup358, suggesting that the CTD does not interact with other nucleoporins.
Assuntos
Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Cristalografia por Raios X , Proteína do Núcleo p24 do HIV/metabolismo , Humanos , Modelos Moleculares , Peptidilprolil Isomerase/metabolismo , Conformação ProteicaRESUMO
Key steps in mRNA export are the nuclear assembly of messenger ribonucleoprotein particles (mRNPs), the translocation of mRNPs through the nuclear pore complex (NPC), and the mRNP remodeling events at the cytoplasmic side of the NPC. Nup358/RanBP2 is a constituent of the cytoplasmic filaments of the NPC specific to higher eukaryotes and provides a multitude of binding sites for the nucleocytoplasmic transport machinery. Here, we present the crystal structure of the Nup358 N-terminal domain (NTD) at 0.95Å resolution. The structure reveals an α-helical domain that harbors three central tetratricopeptide repeats (TPRs), flanked on each side by an additional solvating amphipathic α helix. Overall, the NTD adopts an unusual extended conformation that lacks the characteristic peptide-binding groove observed in canonical TPR domains. Strikingly, the vast majority of the NTD surface exhibits an evolutionarily conserved, positive electrostatic potential, and we demonstrate that the NTD possesses the capability to bind single-stranded RNA in solution. Together, these data suggest that the NTD contributes to mRNP remodeling events at the cytoplasmic face of the NPC.
Assuntos
Chaperonas Moleculares/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , RNA/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Erythrocyte-binding antigen 140 (PfEBA-140) is a critical Plasmodium falciparum erythrocyte invasion ligand that engages glycophorin C on host erythrocytes during malaria infection. The minimal receptor-binding region of PfEBA-140 contains two conserved Duffy binding-like (DBL) domains, a fold unique to Plasmodium species. Here, we present the crystal structure of the receptor-binding region of PfEBA-140 at 2.4 Å resolution. The two-domain binding region is present as a monomer in the asymmetric unit, and the structure reveals novel features in PfEBA-140 that are likely determinants of receptor specificity. Analysis by small-angle x-ray scattering demonstrated that the minimal binding region is monomeric in solution, consistent with the crystal structure. Erythrocyte binding assays showed that the full-length binding region containing the tandem DBL domains is required for erythrocyte engagement, suggesting that both domains contain critical receptor contact sites. The electrostatic surface of PfEBA-140 elucidates a basic patch that constitutes a putative high-affinity binding interface spanning both DBL domains. Mutation of residues within this interface results in severely diminished erythrocyte binding. This study provides insight into the structural basis and mechanism of PfEBA-140 receptor engagement and forms a basis for future studies of this critical interaction. In addition, the solution and crystal structures allow the first identification of likely determinants of erythrocyte receptor specificity for P. falciparum invasion ligands. A complete understanding of the PfEBA-140 erythrocyte invasion pathway will aid in the design of invasion inhibitory therapeutics and vaccines.