RESUMEN
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 â¼110-MDa, â¼1000-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.
Asunto(s)
Núcleo Celular , Poro Nuclear , Humanos , Poro Nuclear/química , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Retículo Endoplásmico , Eucariontes , Membrana Nuclear/metabolismoRESUMEN
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].
Asunto(s)
Citoplasma , Proteínas Fúngicas , Proteínas de Complejo Poro Nuclear , Poro Nuclear , Transporte de ARN , ARN Mensajero , Chaetomium , Microscopía por Crioelectrón , Citoplasma/química , Proteínas Fúngicas/química , Humanos , Chaperonas Moleculares/química , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , ARN Mensajero/metabolismoRESUMEN
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].
Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Proteínas de Saccharomyces cerevisiae , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
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.
Asunto(s)
Proteínas de Complejo Poro Nuclear , Poro Nuclear , Membrana Nuclear , Oocitos , OogénesisRESUMEN
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.
Asunto(s)
Modelos Moleculares , Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Microscopía por Crioelectrón , Cristalografía por Rayos X , Eucariontes/metabolismo , Eucariontes/ultraestructura , Humanos , Poro Nuclear/ultraestructura , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , Subunidades de Proteína , ARN Mensajero/metabolismoRESUMEN
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.
Asunto(s)
Poro Nuclear/química , Poro Nuclear/metabolismo , ARN Mensajero/química , ARN Mensajero/metabolismo , Transporte Activo de Núcleo Celular , Chaetomium/genética , Chaetomium/metabolismo , ARN Helicasas DEAD-box/química , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Modelos Biológicos , Modelos Moleculares , Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ácido Fítico/metabolismo , Transporte de ARN , ARN Mensajero/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/metabolismoRESUMEN
Determining macromolecular structures from X-ray data with resolution worse than 3â Å remains 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 3.0â Å resolution F1-ATPase data set and a 4.5â Å resolution 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.
RESUMEN
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.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Histonas/química , Histonas/metabolismo , Células Mieloides/citología , Células Mieloides/metabolismo , Acetilación , Diferenciación Celular/fisiología , Cromatina/química , Cromatina/metabolismo , Cristalografía por Rayos X , Hematopoyesis/fisiología , Humanos , Modelos Moleculares , Unión Proteica , Dominios Proteicos , Factores de TranscripciónRESUMEN
Eukaryotic ribosome biogenesis requires the nuclear import of â¼80 nascent ribosomal proteins and the elimination of excess amounts by the cellular degradation machinery. Assembly chaperones recognize nascent unassembled ribosomal proteins and transport them together with karyopherins to their nuclear destination. We report the crystal structure of ribosomal protein L4 (RpL4) bound to its dedicated assembly chaperone of L4 (Acl4), revealing extensive interactions sequestering 70 exposed residues of the extended RpL4 loop. The observed molecular recognition fundamentally differs from canonical promiscuous chaperone-substrate interactions. We demonstrate that the eukaryote-specific RpL4 extension harbours overlapping binding sites for Acl4 and the nuclear transport factor Kap104, facilitating its continuous protection from the cellular degradation machinery. Thus, Acl4 serves a dual function to facilitate nuclear import and simultaneously protect unassembled RpL4 from the cellular degradation machinery.
Asunto(s)
Proteínas Ribosómicas/metabolismo , Transporte Activo de Núcleo Celular , Sitios de Unión , Cristalografía por Rayos X , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Unión Proteica , Conformación Proteica , Proteolisis , Proteínas Ribosómicas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinación , beta Carioferinas/química , beta Carioferinas/metabolismoRESUMEN
Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.
Asunto(s)
Redes y Vías Metabólicas , Proteolisis , Proteínas Ribosómicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Eliminación de Gen , Control de Calidad , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Supresoras de Tumor , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Elucidating the structure of the nuclear pore complex (NPC) is a prerequisite for understanding the molecular mechanism of nucleocytoplasmic transport. However, owing to its sheer size and flexibility, the NPC is unapproachable by classical structure determination techniques and requires a joint effort of complementary methods. Whereas bottom-up approaches rely on biochemical interaction studies and crystal-structure determination of NPC components, top-down approaches attempt to determine the structure of the intact NPC in situ. Recently, both approaches have converged, thereby bridging the resolution gap from the higher-order scaffold structure to near-atomic resolution and opening the door for structure-guided experimental interrogations of NPC function.
Asunto(s)
Chaperonas Moleculares/química , Proteínas de Complejo Poro Nuclear/química , Poro Nuclear/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Expresión Génica , Humanos , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/genética , Proteínas de Complejo Poro Nuclear/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Proteínas de Complejo Poro Nuclear/metabolismo , Poro Nuclear/metabolismo , Poro Nuclear/ultraestructura , Mapas de Interacción de Proteínas , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Citoplasma/metabolismo , Tomografía con Microscopio Electrónico , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Datos de Secuencia Molecular , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
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.
Asunto(s)
Chaetomium/ultraestructura , Proteínas Fúngicas/ultraestructura , Proteínas de Complejo Poro Nuclear/ultraestructura , Poro Nuclear/ultraestructura , Proteínas Nucleares/ultraestructura , Secuencia de Aminoácidos , Chaetomium/metabolismo , Proteínas Fúngicas/química , Datos de Secuencia Molecular , Poro Nuclear/metabolismo , Proteínas de Complejo Poro Nuclear/química , Proteínas Nucleares/química , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Eukaryotic ribosome biogenesis requires nuclear import and hierarchical incorporation of â¼80 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 â¼80-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.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Ribosómicas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Proteínas Fúngicas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Proteínas Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/metabolismo , Saccharomyces cerevisiaeRESUMEN
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.
Asunto(s)
Proteínas de Complejo Poro Nuclear/química , Poro Nuclear/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/ultraestructura , Cristalografía por Rayos X , Humanos , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Tubulin protomers undergo an extensive array of post-translational modifications to tailor microtubules to specific tasks. One such modification, the acetylation of lysine 40 of α-tubulin, located in the lumen of microtubules, is associated with stable, long-living microtubule structures. MEC-17 was recently identified as the acetyltransferase that mediates this event. We have determined the crystal structure of the catalytic core of human MEC-17 in complex with its cofactor acetyl-CoA at 1.7Å resolution. The structure reveals that the MEC-17 core adopts a canonical Gcn5-related N-acetyltransferase (GNAT) fold that is decorated with extensive surface loops. An enzymatic analysis of 33 MEC-17 surface mutants identifies hot-spot residues for catalysis and substrate recognition. A large, evolutionarily conserved hydrophobic surface patch that is critical for enzymatic activity is identified, suggesting that specificity is achieved by interactions with the α-tubulin substrate that extend outside of the modified surface loop. An analysis of MEC-17 mutants in Caenorhabditis elegans shows that enzymatic activity is dispensable for touch sensitivity.
Asunto(s)
Acetiltransferasas/química , Acetiltransferasas/metabolismo , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetiltransferasas/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografía por Rayos X , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Proteínas de Microtúbulos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Tubulina (Proteína)/metabolismoRESUMEN
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.
Asunto(s)
Evolución Molecular , Carioferinas/genética , Modelos Moleculares , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/genética , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Calorimetría , Cristalografía por Rayos X , Hibridación Fluorescente in Situ , Carioferinas/química , Mutagénesis Sitio-Dirigida , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
SIRT1 is a NAD(+)-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a ß hairpin structure that complements the ß sheet of the NAD(+)-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD(+)-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.