[ { "id": "authors:vcpvz-cyc44", "collection": "authors", "collection_id": "vcpvz-cyc44", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220916-663648000", "type": "article", "title": "Structure and Function of the Nuclear Pore Complex", "author": [ { "family_name": "Petrovic", "given_name": "Stefan", "orcid": "0000-0002-4979-8696", "clpid": "Petrovic-Stefan" }, { "family_name": "Mobbs", "given_name": "George W.", "orcid": "0000-0003-2405-3345", "clpid": "Mobbs-George-W" }, { "family_name": "Bley", "given_name": "Christopher J.", "orcid": "0000-0003-2301-1290", "clpid": "Bley-Christopher-J" }, { "family_name": "Nie", "given_name": "Si", "orcid": "0000-0002-1347-0656", "clpid": "Nie-Si" }, { "family_name": "Patke", "given_name": "Alina", "orcid": "0000-0003-1628-4282", "clpid": "Patke-Alina" }, { "family_name": "Hoelz", "given_name": "Andr\u00e9", "orcid": "0000-0003-1726-0127", "clpid": "Hoelz-A" } ], "abstract": "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 \u223c110-MDa, \u223c1000-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\u2013pathogen interactions, serving as a paradigm for studying similarly large complexes.", "doi": "10.1101/cshperspect.a041264", "issn": "1943-0264", "publisher": "Cold Spring Harbor Laboratory Press", "publication": "Cold Spring Harbor Perspectives in Biology", "publication_date": "2022-12", "series_number": "12", "volume": "14", "issue": "12", "pages": "Art. No. a041264" }, { "id": "authors:sfg7x-dk730", "collection": "authors", "collection_id": "sfg7x-dk730", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211102-164838429", "type": "article", "title": "Architecture of the linker-scaffold in the nuclear pore", "author": [ { "family_name": "Petrovic", "given_name": "Stefan", "orcid": "0000-0002-4979-8696", "clpid": "Petrovic-Stefan" }, { "family_name": "Samanta", "given_name": "Dipanjan", "orcid": "0000-0001-8920-7312", "clpid": "Samanta-Dipanjan" }, { "family_name": "Perriches", "given_name": "Thibaud", "clpid": "Perriches-Thibaud" }, { "family_name": "Bley", "given_name": "Christopher J.", "orcid": "0000-0003-2301-1290", "clpid": "Bley-Christopher-J" }, { "family_name": "Thierbach", "given_name": "Karsten", "clpid": "Thierbach-Karsten" }, { "family_name": "Brown", "given_name": "Bonnie", "clpid": "Brown-Bonnie" }, { "family_name": "Nie", "given_name": "Si", "orcid": "0000-0002-1347-0656", "clpid": "Nie-Si" }, { "family_name": "Mobbs", "given_name": "George W.", "orcid": "0000-0003-2405-3345", "clpid": "Mobbs-George-W" }, { "family_name": "Stevens", "given_name": "Taylor A.", "orcid": "0000-0002-6232-5316", "clpid": "Stevens-Taylor-A" }, { "family_name": "Liu", "given_name": "Xiaoyu", "orcid": "0000-0002-6594-9677", "clpid": "Liu-Xiaoyu" }, { "family_name": "Pinton Tomaleri", "given_name": "Giovani", "orcid": "0000-0001-9661-6480", "clpid": "Pinton-Tomaleri-Giovani" }, { "family_name": "Schaus", "given_name": "Lucas", "orcid": "0000-0002-6094-7402", "clpid": "Schaus-Lucas" }, { "family_name": "Hoelz", "given_name": "Andr\u00e9", "orcid": "0000-0003-1726-0127", "clpid": "Hoelz-A" } ], "abstract": "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-\u00c5-wide and ~750-\u00c5-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. \n\nRATIONALE. Although the arrangement of most scaffold nucleoporins in the NPC's symmetric core was determined by quantitative docking of crystal structures into cryo\u2013electron 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 \u00c5. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo\u2013electron 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. \n \nRESULTS. 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\u2013shaped keystones of two evolutionarily conserved hetero\u2011octameric 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 structure\u2011guided functional analysis in Saccharomyces cerevisiae, we confirmed the robustness of linker\u2011scaffold 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 linker\u2011scaffold 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. \n\nCONCLUSION. 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 linker\u2011scaffold 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 NPC\u2011associated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins.", "doi": "10.1126/science.abm9798", "pmcid": "PMC9867570", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2022-06-10", "series_number": "6598", "volume": "376", "issue": "6598", "pages": "Art. No. abm9798" }, { "id": "authors:36v8f-0a053", "collection": "authors", "collection_id": "36v8f-0a053", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20211102-155046319", "type": "article", "title": "Architecture of the cytoplasmic face of the nuclear pore", "author": [ { "family_name": "Bley", "given_name": "Christopher J.", "orcid": "0000-0003-2301-1290", "clpid": "Bley-Christopher-J" }, { "family_name": "Nie", "given_name": "Si", "orcid": "0000-0002-1347-0656", "clpid": "Nie-Si" }, { "family_name": "Mobbs", "given_name": "George W.", "orcid": "0000-0003-2405-3345", "clpid": "Mobbs-George-W" }, { "family_name": "Petrovic", "given_name": "Stefan", "orcid": "0000-0002-4979-8696", "clpid": "Petrovic-Stefan" }, { "family_name": "Gres", "given_name": "Anna T.", "orcid": "0000-0002-6915-5903", "clpid": "Gres-Anna-T" }, { "family_name": "Liu", "given_name": "Xiaoyu", "orcid": "0000-0002-6594-9677", "clpid": "Liu-Xiaoyu" }, { "family_name": "Mukherjee", "given_name": "Somnath", "orcid": "0000-0001-5447-4496", "clpid": "Mukherjee-Somnath" }, { "family_name": "Harvey", "given_name": "Sho", "orcid": "0000-0002-1742-4550", "clpid": "Harvey-Sho" }, { "family_name": "Huber", "given_name": "Ferdinand M.", "orcid": "0000-0001-8647-6971", "clpid": "Huber-Ferdinand-M" }, { "family_name": "Lin", "given_name": "Daniel H.", "orcid": "0000-0002-1393-3374", "clpid": "Lin-Daniel-H" }, { "family_name": "Brown", "given_name": "Bonnie", "clpid": "Brown-Bonnie" }, { "family_name": "Tang", "given_name": "Aaron W.", "orcid": "0000-0001-8891-6205", "clpid": "Tang-Aaron-W" }, { "family_name": "Rundlet", "given_name": "Emily J.", "orcid": "0000-0003-4041-6206", "clpid": "Rundlet-Emily-J" }, { "family_name": "Correia", "given_name": "Ana R.", "clpid": "Correia-Ana-R" }, { "family_name": "Chen", "given_name": "Shane", "clpid": "Che-Shane" }, { "family_name": "Regmi", "given_name": "Saroj G.", "orcid": "0000-0002-6705-3997", "clpid": "Regmi-Saroj-G" }, { "family_name": "Stevens", "given_name": "Taylor A.", "orcid": "0000-0002-6232-5316", "clpid": "Stevens-Taylor-A" }, { "family_name": "Jette", "given_name": "Claudia A.", "orcid": "0000-0002-5085-8027", "clpid": "Jette-Claudia-A" }, { "family_name": "Dasso", "given_name": "Mary", "orcid": "0000-0002-5410-1371", "clpid": "Dasso-Mary" }, { "family_name": "Patke", "given_name": "Alina", "orcid": "0000-0003-1628-4282", "clpid": "Patke-Alina" }, { "family_name": "Palazzo", "given_name": "Alexander F.", "orcid": "0000-0002-9700-1995", "clpid": "Palazzo-Alexander-F" }, { "family_name": "Kossiakoff", "given_name": "Anthony A.", "orcid": "0000-0003-3174-9359", "clpid": "Kossiakoff-Anthony-A" }, { "family_name": "Hoelz", "given_name": "Andr\u00e9", "orcid": "0000-0003-1726-0127", "clpid": "Hoelz-A" } ], "abstract": "The nuclear pore complex (NPC) is the sole bidirectional gateway for nucleocytoplasmic transport. Despite recent progress in elucidating the NPC symmetric core architecture, the asymmetrically decorated cytoplasmic face, essential for messenger RNA (mRNA) export and a hotspot for nucleoporin-associated diseases, has remained elusive. Here we report a composite structure of the human cytoplasmic face obtained by combining biochemical reconstitution, crystal structure determination, docking into cryo\u2013electron tomographic reconstructions, and physiological validation. Whereas species-specific motifs anchor an evolutionarily conserved ~540-kilodalton heterohexameric cytoplasmic filament nucleoporin complex above the central transport channel, attachment of the NUP358 pentameric bundles depends on the double-ring arrangement of the coat nucleoporin complex. Our composite structure and its predictive power provide a rich foundation for elucidating the molecular basis of mRNA export and nucleoporin diseases.", "doi": "10.1126/science.abm9129", "pmcid": "PMC9348906", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2022-06-10", "series_number": "6598", "volume": "376", "issue": "6598", "pages": "Art. No. abm9129" }, { "id": "authors:5mq87-nwx44", "collection": "authors", "collection_id": "5mq87-nwx44", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20220329-4699029", "type": "article", "title": "Molecular basis of specificity and deamidation of eIF4A by Burkholderia Lethal Factor 1", "author": [ { "family_name": "Mobbs", "given_name": "George W.", "orcid": "0000-0003-2405-3345", "clpid": "Mobbs-George-W" }, { "family_name": "Aziz", "given_name": "Adli A.", "orcid": "0000-0002-2665-2467", "clpid": "Aziz-Adli-A" }, { "family_name": "Dix", "given_name": "Samuel R.", "orcid": "0000-0002-6907-1435", "clpid": "Dix-Samuel-R" }, { "family_name": "Blackburn", "given_name": "G. M.", "orcid": "0000-0002-3941-0459", "clpid": "Blackburn-George-Michael" }, { "family_name": "Sedelnikova", "given_name": "Sveta E.", "orcid": "0000-0001-5174-6302", "clpid": "Sedelnikova-Svetlana-E" }, { "family_name": "Minshull", "given_name": "Thomas C.", "clpid": "Minshull-Thomas-C" }, { "family_name": "Dickman", "given_name": "Mark J.", "orcid": "0000-0002-9236-0788", "clpid": "Dickman-Mark-J" }, { "family_name": "Baker", "given_name": "Patrick J.", "orcid": "0000-0003-1995-5643", "clpid": "Baker-Patrick-J" }, { "family_name": "Nathan", "given_name": "Sheila", "orcid": "0000-0002-2132-2346", "clpid": "Nathan-Sheila" }, { "family_name": "Firdaus Raih", "given_name": "Mohd", "orcid": "0000-0003-4275-4663", "clpid": "Firdaus-Raih-Mohd" }, { "family_name": "Rice", "given_name": "David W.", "orcid": "0000-0002-7811-0539", "clpid": "Rice-David-W" } ], "abstract": "AbstractBurkholderiapseudomallei lethal factor 1 (BLF1) exhibits site-specific glutamine deamidase activity against the eukaryotic RNA helicase, eIF4A, thereby blocking mammalian protein synthesis. The structure of a complex between BLF1 C94S and human eIF4A shows that the toxin binds in the cleft between the two RecA-like eIF4A domains forming interactions with residues from both and with the scissile amide of the target glutamine, Gln339, adjacent to the toxin active site. The RecA-like domains adopt a radically twisted orientation compared to other eIF4A structures and the nature and position of conserved residues suggests this may represent a conformation associated with RNA binding. Comparison of the catalytic site of BLF1 with other deamidases and cysteine proteases reveals that they fall into two classes, related by pseudosymmetry, that present either the re or si faces of the target amide/peptide to the nucleophilic sulfur, highlighting constraints in the convergent evolution of their Cys-His active sites.", "doi": "10.1038/s42003-022-03186-2", "pmcid": "PMC8960835", "issn": "2399-3642", "publisher": "Springer Science and Business Media LLC", "publication": "Communications Biology", "publication_date": "2022-03-28", "volume": "5", "pages": "Art. No. 272" }, { "id": "authors:0ddes-yya74", "collection": "authors", "collection_id": "0ddes-yya74", "cite_using_url": "https://resolver.caltech.edu/CaltechAUTHORS:20160414-150533638", "type": "article", "title": "Architecture of the symmetric core of the nuclear pore", "author": [ { "family_name": "Lin", "given_name": "Daniel H.", "clpid": "Lin-Daniel-H" }, { "family_name": "Stuwe", "given_name": "Tobias", "clpid": "Stuwe-Tobias" }, { "family_name": "Schilbach", "given_name": "Sandra", "orcid": "0000-0001-9071-8515", "clpid": "Schilbach-Sandra" }, { "family_name": "Rundlet", "given_name": "Emily J.", "clpid": "Rundlet-Emily-J" }, { "family_name": "Perriches", "given_name": "Thibaud", "clpid": "Perriches-Thibaud" }, { "family_name": "Mobbs", "given_name": "George", "orcid": "0000-0003-2405-3345", "clpid": "Mobbs-George-W" }, { "family_name": "Fan", "given_name": "Yanbin", "clpid": "Fan-Yanbin" }, { "family_name": "Thierbach", "given_name": "Karsten", "clpid": "Thierbach-Karsten" }, { "family_name": "Huber", "given_name": "Ferdinand M.", "orcid": "0000-0001-8647-6971", "clpid": "Huber-Ferdinand-M" }, { "family_name": "Collins", "given_name": "Leslie N.", "clpid": "Collins-Leslie-N" }, { "family_name": "Davenport", "given_name": "Andrew M.", "clpid": "Davenport-Andrew-M" }, { "family_name": "Jeon", "given_name": "Young E.", "clpid": "Jeon-Young-E" }, { "family_name": "Hoelz", "given_name": "Andr\u00e9", "orcid": "0000-0003-1726-0127", "clpid": "Hoelz-A" } ], "abstract": "INTRODUCTION: The nuclear pore complex (NPC) is the primary gateway for the transport of macromolecules between the nucleus and cytoplasm, serving as both a critical mediator and regulator of gene expression. NPCs are very large (~120 MDa) macromolecular machines embedded in the nuclear envelope, each containing ~1000 protein subunits, termed nucleoporins. Despite substantial progress in visualizing the overall shape of the NPC by means of cryoelectron tomography (cryo-ET) and in determining atomic-resolution crystal structures of nucleoporins, the molecular architecture of the assembled NPC has thus far remained poorly understood, hindering the design of mechanistic studies that could investigate its many roles in cell biology. \n\nRATIONALE: Existing cryo-ET reconstructions of the NPC are too low in resolution to allow for de novo structure determination of the NPC or unbiased docking of nucleoporin fragment crystal structures. We sought to bridge this resolution gap by first defining the interaction network of the NPC, focusing on the evolutionarily conserved symmetric core. We developed protocols to reconstitute NPC protomers from purified recombinant proteins, which enabled the generation of a high-resolution biochemical interaction map of the NPC symmetric core. We next determined high-resolution crystal structures of key nucleoporin interactions, providing spatial restraints for their relative orientation. By superposing crystal structures that overlapped in sequence, we generated accurate full-length structures of the large scaffold nucleoporins. Lastly, we used sequential unbiased searches, supported by the biochemical data, to place the nucleoporin crystal structures into a previously determined cryo-ET reconstruction of the intact human NPC, thus generating a composite structure of the entire NPC symmetric core. \n\nRESULTS: Our analysis revealed that the inner and outer rings of the NPC use disparate mechanisms of interaction. Whereas the structured coat nucleoporins of the outer ring form extensive surface contacts, the scaffold proteins of the inner ring are bridged by flexible sequences in linker nucleoporins. Our composite structure revealed a defined spoke architecture in which each of the eight spokes spans the nuclear envelope, with limited cross-spoke interactions. Most nucleoporins are present in 32 copies, with the exceptions of Nup170 and Nup188, which are present in 48 and 16 copies, respectively. Lastly, we observed the arrangement of the channel nucleoporins, which orient their N termini into two 16-membered rings, thus ensuring that their N-terminal FG repeats project evenly into the central transport channel. \n\nCONCLUSION: Our composite structure of the NPC symmetric core can be used as a platform for the rational design of experiments to investigate NPC structure and function. Each nucleoporin occupies multiple distinct biochemical environments, explaining how such a large macromolecular complex can be assembled from a relatively small number of genes. Our integrated, bottom-up approach provides a paradigm for the biochemical and structural characterization of similarly large biological mega-assemblies.", "doi": "10.1126/science.aaf1015", "pmcid": "PMC5207208", "issn": "0036-8075", "publisher": "American Association for the Advancement of Science", "publication": "Science", "publication_date": "2016-04-15", "series_number": "6283", "volume": "352", "issue": "6283", "pages": "Art. No. aaf1015" } ]