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1.
Annu Rev Biochem ; 88: 725-783, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30883195

RESUMO

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 (∼1,000 protein subunits, ∼110 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/metabolismo
2.
Annu Rev Biochem ; 80: 613-43, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21495847

RESUMO

In eukaryotic cells, the spatial segregation of replication and transcription in the nucleus and translation in the cytoplasm imposes the requirement of transporting thousands of macromolecules between these two compartments. Nuclear pore complexes (NPCs) are the sole gateways that facilitate this macromolecular exchange across the nuclear envelope with the help of soluble transport receptors. Whereas the mobile transport machinery is reasonably well understood at the atomic level, a commensurate structural characterization of the NPC has only begun in the past few years. Here, we describe the recent progress toward the elucidation of the atomic structure of the NPC, highlight emerging concepts of its underlying architecture, and discuss key outstanding questions and challenges. The applied structure determination as well as the described design principles of the NPC may serve as paradigms for other macromolecular assemblies.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Poro Nuclear/ultraestrutura , Conformação Proteica , Animais , Cristalografia por Raios X , Evolução Molecular , Humanos , Microscopia Eletrônica , Modelos Moleculares , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , beta Carioferinas/química , beta Carioferinas/metabolismo
3.
Trends Biochem Sci ; 45(4): 278-280, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32169173

RESUMO

Oocytes stockpile nuclear pore complexes (NPCs) in cytoplasmic membrane sheets called annulate lamellae (AL) in preparation for rapid cell cycles during embryogenesis. Recently, Hampoelz et al. reported that AL-NPC assembly depends on the coordinated formation, transport, and interaction of biomolecular condensates containing distinct sets of nucleoporins.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Membrana Nuclear , Oócitos , Oogênese
4.
Mol Cell ; 58(5): 854-62, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25936803

RESUMO

Eukaryotic ribosome biogenesis requires nuclear import and hierarchical incorporation of ∼80 ribosomal proteins (RPs) into the ribosomal RNA core. In contrast to prokaryotes, many eukaryotic RPs possess long extensions that interdigitate in the mature ribosome. RpL4 is a prime example, with an âˆ¼80-residue-long surface extension of unknown function. Here, we identify assembly chaperone Acl4 that initially binds the universally conserved internal loop of newly synthesized RpL4 via its superhelical TPR domain, thereby restricting RpL4 loop insertion at its cognate nascent rRNA site. RpL4 release from Acl4 is orchestrated with pre-ribosome assembly, during which the eukaryote-specific RpL4 extension makes several distinct interactions with the 60S surface, including a co-evolved site on neighboring RpL18. Consequently, mutational inactivation of this contact site, on either RpL4 or RpL18, impairs RpL4-Acl4 disassembly and RpL4 pre-ribosome incorporation. We propose that hierarchical ribosome assembly can be achieved by eukaryotic RP extensions and dedicated assembly chaperones.


Assuntos
Proteínas Fúngicas/química , Proteínas Ribossômicas/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae
5.
Proc Natl Acad Sci U S A ; 114(23): 6016-6021, 2017 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-28533407

RESUMO

Double plant homeodomain finger 2 (DPF2) is a highly evolutionarily conserved member of the d4 protein family that is ubiquitously expressed in human tissues and was recently shown to inhibit the myeloid differentiation of hematopoietic stem/progenitor and acute myelogenous leukemia cells. Here, we present the crystal structure of the tandem plant homeodomain finger domain of human DPF2 at 1.6-Å resolution. We show that DPF2 interacts with the acetylated tails of both histones 3 and 4 via bipartite binding pockets on the DPF2 surface. Blocking these interactions through targeted mutagenesis of DPF2 abolishes its recruitment to target chromatin regions as well as its ability to prevent myeloid differentiation in vivo. Our findings suggest that the histone binding of DPF2 plays an important regulatory role in the transcriptional program that drives myeloid differentiation.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/química , Histonas/metabolismo , Células Mieloides/citologia , Células Mieloides/metabolismo , Acetilação , Diferenciação Celular/fisiologia , Cromatina/química , Cromatina/metabolismo , Cristalografia por Raios X , Hematopoese/fisiologia , Humanos , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Fatores de Transcrição
6.
Proc Natl Acad Sci U S A ; 111(7): 2530-5, 2014 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-24505056

RESUMO

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 ∼110-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 karyopherin•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/metabolismo
7.
Mol Cell ; 32(6): 815-26, 2008 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-19111661

RESUMO

We recently proposed a cylindrical coat for the nuclear pore membrane in the nuclear pore complex (NPC). This scaffold is generated by multiple copies of seven nucleoporins. Here, we report three crystal structures of the nucleoporin pair Seh1*Nup85, which is part of the coat cylinder. The Seh1*Nup85 assembly bears resemblance in its shape and dimensions to that of another nucleoporin pair, Sec13*Nup145C. Furthermore, the Seh1*Nup85 structures reveal a hinge motion that may facilitate conformational changes in the NPC during import of integral membrane proteins and/or during nucleocytoplasmic transport. We propose that Seh1*Nup85 and Sec13*Nup145C form 16 alternating, vertical rods that are horizontally linked by the three remaining nucleoporins of the coat cylinder. Shared architectural and mechanistic principles with the COPII coat indicate a common evolutionary origin and support the notion that the NPC coat represents another class of membrane coats.


Assuntos
Poro Nuclear/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Maleabilidade , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Soluções , Propriedades de Superfície
8.
Proc Natl Acad Sci U S A ; 108(40): 16571-6, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21930948

RESUMO

So far, only a few of the interactions between the ≈ 30 nucleoporins comprising the modular structure of the nuclear pore complex have been defined at atomic resolution. Here we report the crystal structure, at 2.6 Å resolution, of a heterotrimeric complex, composed of fragments of three cytoplasmically oriented nucleoporins of yeast: Nup82, Nup116, and Nup159. Our data show that the Nup82 fragment, representing more than the N-terminal half of the molecule, folds into an extensively decorated, seven-bladed ß-propeller that forms the centerpiece of this heterotrimeric complex and anchors both a C-terminal fragment of Nup116 and the C-terminal tail of Nup159. Binding between Nup116 and Nup82 is mutually reinforced via two loops, one emanating from the Nup82 ß-propeller and the other one from the ß-sandwich fold of Nup116, each contacting binding pockets in their counterparts. The Nup82-Nup159 interaction occurs through an amphipathic α-helix of Nup159, which is cradled in a large hydrophobic groove that is generated from several large surface decorations of the Nup82 ß-propeller. Although Nup159 and Nup116 fragments bind to the Nup82 ß-propeller in close vicinity, there are no direct contacts between them, consistent with the noncooperative binding that was detected biochemically. Extensive mutagenesis delineated hot-spot residues for these interactions. We also showed that the Nup82 ß-propeller binds to other yeast Nup116 family members, Nup145N, Nup100 and to the mammalian homolog, Nup98. Notably, each of the three nucleoporins contains additional nuclear pore complex binding sites, distinct from those that were defined here in the heterotrimeric Nup82•Nup159•Nup116 complex.


Assuntos
Modelos Moleculares , Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Conformação Proteica , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/química , Cristalografia por Raios X , Mutagênese Sítio-Dirigida , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Dobramento de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
9.
Proc Natl Acad Sci U S A ; 107(25): 11271-6, 2010 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-20534429

RESUMO

The heptameric coatomer complex forms the protein shell of membrane-bound vesicles that are involved in transport from the Golgi to the endoplasmatic reticulum and in intraGolgi trafficking. The heptamer can be dissected into a heterotetrameric F-subcomplex, which displays similarities to the adapter complex of the "inner" coat in clathrin-coated vesicles, and a heterotrimeric B-subcomplex, which is believed to form an "outer" coat with a morphology distinct from that of clathrin-coated vesicles. We have determined the crystal structure of the complex between the C-terminal domain (CTD) of alpha-COP and full-length epsilon-COP, two components of the B-subcomplex, at a 2.9 A resolution. The alpha-COP(CTD) x epsilon-COP heterodimer forms a rod-shaped structure, in which epsilon-COP adopts a tetratricopeptide repeat (TPR) fold that deviates substantially from the canonical superhelical conformation. The alpha-COP CTD adopts a U-shaped architecture that complements the TPR fold of epsilon-COP. The epsilon-COP TPRs form a circular bracelet that wraps around a protruding beta-hairpin of the alpha-COP CTD, thus interlocking the two proteins. The alpha-COP(CTD) x epsilon-COP complex forms heterodimers in solution, and we demonstrate biochemically that the heterodimer directly interacts with the Dsl1 tethering complex. These data suggest that the heterodimer is exposed on COPI vesicles, while the remaining part of the B-subcomplex oligomerizes underneath into a cage.


Assuntos
Complexo I de Proteína do Envoltório/química , Proteína Coatomer/química , Saccharomyces cerevisiae/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Membrana Celular/metabolismo , Cristalografia por Raios X/métodos , Dimerização , Escherichia coli/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Sequências Repetitivas de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido
10.
Proc Natl Acad Sci U S A ; 107(23): 10406-11, 2010 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-20498086

RESUMO

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 A resolution. Rae1 forms a seven-bladed beta-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an approximately 50-A-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 beta-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1*Nup98(GLEBS) surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1*Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.


Assuntos
Proteínas Associadas à Matriz Nuclear/química , Proteínas Associadas à Matriz Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte de RNA , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Relação Estrutura-Atividade , Propriedades de Superfície
11.
Proc Natl Acad Sci U S A ; 106(34): 14281-6, 2009 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-19706512

RESUMO

The Nup84 complex constitutes a key building block in the nuclear pore complex (NPC). Here we present the crystal structure of one of its 7 components, Nup120, which reveals a beta propeller and an alpha-helical domain representing a novel fold. We discovered a previously unidentified interaction of Nup120 with Nup133 and confirmed the physiological relevance in vivo. As mapping of the individual components in the Nup84 complex places Nup120 and Nup133 at opposite ends of the heptamer, our findings indicate a head-to-tail arrangement of elongated Nup84 complexes into a ring structure, consistent with a fence-like coat for the nuclear pore membrane. The attachment site for Nup133 lies at the very end of an extended unstructured region, which allows for flexibility in the diameter of the Nup84 complex ring. These results illuminate important roles of terminal unstructured segments in nucleoporins for the architecture, function, and assembly of the NPC.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Cristalização , Teste de Complementação Genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Difração de Raios X
12.
Proc Natl Acad Sci U S A ; 106(9): 3089-94, 2009 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-19208808

RESUMO

Key steps in the export of mRNA from the nucleus to the cytoplasm are the transport through the nuclear pore complex (NPC) and the subsequent remodeling of messenger RNA-protein (mRNP) complexes that occurs at the cytoplasmic side of the NPC. Crucial for these events is the recruitment of the DEAD-box helicase Ddx19 to the cytoplasmic filaments of the NPC that is mediated by the nucleoporin Nup214. Here, we present the crystal structure of the Nup214 N-terminal domain in complex with Ddx19 in its ADP-bound state at 2.5 A resolution. Strikingly, the interaction surfaces are not only evolutionarily conserved but also exhibit strongly opposing surface potentials, with the helicase surface being positively and the Nup214 surface being negatively charged. We speculate that the positively charged surface of the interacting ADP-helicase binds competitively to a segment of mRNA of a linearized mRNP, passing through the NPC on its way to the cytoplasm. As a result, the ADP-helicase would dissociate from Nup214 and replace a single bound protein from the mRNA. One cycle of protein replacement would be accompanied, cooperatively, by nucleotide exchange, ATP hydrolysis, release of the ADP-helicase from mRNA and its rebinding to Nup214. Repeat of these cycles would remove proteins from a mRNP, one at a time, akin to a ratchet mechanism for mRNA export.


Assuntos
RNA Helicases DEAD-box/química , RNA Helicases DEAD-box/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Ligação Proteica , Estrutura Quaternária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Propriedades de Superfície
13.
Proc Natl Acad Sci U S A ; 106(42): 17693-8, 2009 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-19805193

RESUMO

The heptameric Nup84 complex constitutes an evolutionarily conserved building block of the nuclear pore complex. Here, we present the crystal structure of the heterotrimeric Sec13 x Nup145C x Nup84 complex, the centerpiece of the heptamer, at 3.2-A resolution. Nup84 forms a U-shaped alpha-helical solenoid domain, topologically similar to two other members of the heptamer, Nup145C and Nup85. The interaction between Nup84 and Nup145C is mediated via a hydrophobic interface located in the kink regions of the two solenoids that is reinforced by additional interactions of two long Nup84 loops. The Nup84 binding site partially overlaps with the homo-dimerization interface of Nup145C, suggesting competing binding events. Fitting of the elongated Z-shaped heterotrimer into electron microscopy (EM) envelopes of the heptamer indicates that structural changes occur at the Nup145C x Nup84 interface. Docking the crystal structures of all heptamer components into the EM envelope constitutes a major advance toward the completion of the structural characterization of the Nup84 complex.


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/química , Poro Nuclear/química , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , Ligação Competitiva , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/química
14.
Artigo em Inglês | MEDLINE | ID: mdl-36096637

RESUMO

The nucleus, a genome-containing organelle eponymous of eukaryotes, is enclosed by a double membrane continuous with the endoplasmic reticulum. The nuclear pore complex (NPC) is an ∼110-MDa, ∼1000-protein channel that selectively transports macromolecules across the nuclear envelope and thus plays a central role in the regulated flow of genetic information from transcription to translation. Its size, complexity, and flexibility have hindered determination of atomistic structures of intact NPCs. Recent studies have overcome these hurdles by combining biochemical reconstitution and docking of high-resolution structures of NPC subcomplexes into cryo-electron tomographic reconstructions with biochemical and physiological validation. Here, we provide an overview of the near-atomic composite structure of the human NPC, a milestone toward unlocking a molecular understanding of mRNA export, NPC-associated diseases, and viral host-pathogen interactions, serving as a paradigm for studying similarly large complexes.


Assuntos
Núcleo Celular , Poro Nuclear , Humanos , Poro Nuclear/química , Poro Nuclear/metabolismo , Transporte Ativo do Núcleo Celular , Retículo Endoplasmático , Eucariotos , Membrana Nuclear/metabolismo
15.
Science ; 376(6598): eabm9798, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679425

RESUMO

INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC's eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC's symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC's symmetric core was determined by quantitative docking of crystal structures into cryo-electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC's inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo-electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC's integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark-shaped keystones of two evolutionarily conserved hetero­octameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structure­guided functional analysis in Saccharomyces cerevisiae, we confirmed the robustness of linker­scaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and S. cerevisiae cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linker­scaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the S. cerevisiae and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linker­scaffold establishes the NPC's integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPC­associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. [Figure: see text].


Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Proteínas de Saccharomyces cerevisiae , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química
16.
Science ; 376(6598): eabm9129, 2022 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-35679405

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/metabolismo
17.
Nat Struct Mol Biol ; 13(7): 626-32, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16799558

RESUMO

The reversible methylation of specific lysine residues in histone tails is crucial in epigenetic gene regulation. LSD1, the first known lysine-specific demethylase, selectively removes monomethyl and dimethyl, but not trimethyl modifications of Lys4 or Lys9 of histone-3. Here, we present the crystal structure of LSD1 at 2.9-A resolution. LSD1 forms a highly asymmetric, closely packed domain structure from which a long helical 'tower' domain protrudes. The active site cavity is spacious enough to accommodate several residues of the histone tail substrate, but does not appear capable of recognizing the different methylation states of the substrate lysine. This supports the hypothesis that trimethylated lysine is chemically rather than sterically discriminated. We present a biochemical analysis of LSD1 mutants that identifies crucial residues in the active site cavity and shows the importance of the SWIRM and tower domains for catalysis.


Assuntos
Oxirredutases N-Desmetilantes/química , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Histona Desmetilases , Histonas/metabolismo , Humanos , Lisina/metabolismo , Modelos Moleculares , Mutagênese , Oxirredutases N-Desmetilantes/isolamento & purificação , Conformação Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes/química
18.
Proc Natl Acad Sci U S A ; 105(34): 12236-41, 2008 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-18719104

RESUMO

Rtt109 is a protein acetyltransferase (PAT) that is responsible for the acetylation of lysine-56 of histone 3 (H3K56) in yeast. H3K56 acetylation has been implicated in the weakening of the interaction between the histone core and the surrounding DNA in the nucleosomal particle. Rtt109, in cooperation with various histone chaperones, promotes genomic stability and is required for resistance to DNA damaging agents. Here, we present the crystal structure of Rtt109 in complex with acetyl-CoA at a 2.0-A resolution. Rtt109 consists of a core PAT domain, which binds the acetyl-CoA cofactor. A second domain, the activation domain, is tightly associated with the PAT domain. Autoacetylation of lysine-290 within the activation domain is required for stabilizing the interaction between the two domains and is essential for catalysis. Biochemical analysis demonstrates the requirement of a loop within the PAT domain for the binding of the histone chaperone Vps75, and mutational analysis identifies key residues for catalysis. We propose a model in which the autoacetylation of Rtt109 is crucial for the regulation of its catalytic activity.


Assuntos
Acetilcoenzima A/química , Histona Acetiltransferases/química , Homeostase , Proteínas de Saccharomyces cerevisiae/química , Acetilcoenzima A/metabolismo , Acetilação , Catálise , Cristalografia por Raios X , Estabilidade Enzimática , Histona Acetiltransferases/metabolismo , Histona Acetiltransferases/fisiologia , Chaperonas Moleculares/metabolismo , Mutagênese Sítio-Dirigida , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
19.
Nat Commun ; 9(1): 2319, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899397

RESUMO

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 Nxf1•Nxt1 from mRNAs. Here, we report crystal structures of Gle1•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 Gle1•Nup42•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/metabolismo
20.
IUCrJ ; 5(Pt 2): 166-171, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29765606

RESUMO

Determining macromolecular structures from X-ray data with resolution worse than 3 Šremains a challenge. Even if a related starting model is available, its incompleteness or its bias together with a low observation-to-parameter ratio can render the process unsuccessful or very time-consuming. Yet, many biologically important macromolecules, especially large macromolecular assemblies, membrane proteins and receptors, tend to provide crystals that diffract to low resolution. A new algorithm to tackle this problem is presented that uses a multivariate function to simultaneously exploit information from both an initial partial model and low-resolution single-wavelength anomalous diffraction data. The new approach has been used for six challenging structure determinations, including the crystal structures of membrane proteins and macromolecular complexes that have evaded experts using other methods, and large structures from a 3.0 Šresolution F1-ATPase data set and a 4.5 Šresolution SecYEG-SecA complex data set. All of the models were automatically built by the method to Rfree values of between 28.9 and 39.9% and were free from the initial model bias.

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