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1. X-ray Structure of the Human Karyopherin RanBP5, an Essential Factor for Influenza Polymerase Nuclear Trafficking

Author

Thibaut Crépin, Bruno Da Costa, Christopher Swale, Imre Berger, Andrew A. McCarthy, Frederic Garzoni, Bernard Delmas, Rob W. H. Ruigrok, Laura Sedano, Christoph Bieniossek, Institut de biologie structurale (IBS - UMR 5075), Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), European Molecular Biology Laboratory [Grenoble] (EMBL), Virologie et Immunologie Moléculaires (VIM (UR 0892)), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ)-Université Paris-Saclay-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory (EMBL Grenoble), Max Planck-Bristol Centre for Minimal Biology, F. Hoffmann-La Roche [Basel], Hofmann-La Roche pRED external collaboration programme, ANR-14-CE09-0017,RNAP-IAV,Interactions protéine-protéine et protéine-ARN au sein du complexe réplicatif du virus de la grippe de type A(2014), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 279039,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,COMPLEXINC(2011), Université Paris-Saclay, Centre Lillois d’Études et de Recherches Sociologiques et Économiques - UMR 8019 (CLERSÉ), and Université de Lille-Centre National de la Recherche Scientifique (CNRS)

Subjects
Gene isoform, Models, Molecular, Subfamily, Protein Conformation, Protein combining, medicine.disease_cause, Crystallography, X-Ray, 03 medical and health sciences, Viral Proteins, 0302 clinical medicine, Structural Biology, Influenza A virus, medicine, Humans, Point Mutation, Molecular Biology, Polymerase, 030304 developmental biology, Karyopherin, chemistry.chemical_classification, Cell Nucleus, human karyopherin, 0303 health sciences, Binding Sites, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Point mutation, RNA-Dependent RNA Polymerase, beta Karyopherins, influenza polymerase assembly, Cell biology, Molecular Docking Simulation, Protein Transport, Docking (molecular), PA-PB1 sub-complex nuclear import, biology.protein, NLS-binding site, host–pathogen interaction, 030217 neurology & neurosurgery, Protein Binding
Abstract

International audience; Here, we describe the crystal structures of two distinct isoforms of ligand-free human karyopherin RanBP5 and investigate its global propensity to interact with influenza A virus polymerase. Our results confirm the general architecture and mechanism of the IMB3 karyopherin-β subfamily whilst also highlighting differences with the yeast orthologue Kap121p. Moreover, our results provide insight into the structural flexibility of β-importins in the unbound state. Based on docking of a nuclear localisation sequence, point mutations were designed, which suppress influenza PA-PB1 subcomplex binding to RanBP5 in a binary protein complementation assay.

Published
2020

2. Synthetic self-assembling ADDomer platform for highly efficient vaccination by genetically encoded multiepitope display

Author

Véronique Josserand, Céline Terrat, Matthew Williams, Pascal Fender, Frederic Garzoni, Laurence Chaperot, Fruzsina Rabi, Emilie Stermann, Christopher J. Woods, Bernard Verrier, Joshua C. Bufton, Phil Bates, Gerardo Viedma, Mélanie Guidetti, Imre Berger, Charles Vragniau, Christiane Schaffitzel, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), University of Bristol [Bristol], Imophoron Ltd, Laboratoire de Biologie Tissulaire et d'ingénierie Thérapeutique UMR 5305 (LBTI), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Institute for Advanced Biosciences / Institut pour l'Avancée des Biosciences (Grenoble) (IAB), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Oracle Cloud Development Centre, Institut de biologie et chimie des protéines [Lyon] (IBCP), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Max Planck-Bristol Centre for Minimal Biology, COIFFIER, Celine, Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire [Grenoble] (CHU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Etablissement français du sang - Auvergne-Rhône-Alpes (EFS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes (UGA), ANIMAGE, Rhône-Alpes Genopole, University of Cambridge [UK] (CAM), Laboratoire recherche et développement, EFS, Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory (EMBL Grenoble), and European Molecular Biology Laboratory [Grenoble] (EMBL)

Subjects
Models, Molecular, Scaffold, minimal biology, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Protein Conformation, Computer science, viruses, advanced computing, Epitope, Epitopes, Synthetic biology, 0302 clinical medicine, Virus-like particle, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Structural Biology, Nanotechnology, ComputingMilieux_MISCELLANEOUS, Research Articles, Vaccines, Synthetic, 0303 health sciences, Multidisciplinary, Protein therapeutics, Vaccination, SciAdv r-articles, 3. Good health, Vaccinology, Nanomedicine, 030220 oncology & carcinogenesis, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Synthetic Biology, Genetic Engineering, Research Article, BrisSynBio, Computational biology, Communicable Diseases, Biodesign, Adenoviridae, Structure-Activity Relationship, Viral Proteins, 03 medical and health sciences, [SDV.IMM.VAC] Life Sciences [q-bio]/Immunology/Vaccinology, Self assembling, Humans, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, 030304 developmental biology, Bristol BioDesign Institute, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, [SDV.IMM.IMM]Life Sciences [q-bio]/Immunology/Immunotherapy, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, SYNTHETIC BIOLOGY, [CHIM.POLY]Chemical Sciences/Polymers, [SDV.SP.PG]Life Sciences [q-bio]/Pharmaceutical sciences/Galenic pharmacology, Infectious disease (medical specialty), Communicable Disease Control, [SDV.IMM.VAC]Life Sciences [q-bio]/Immunology/Vaccinology, Vaccine, Epitope Mapping
Abstract

ADDomer is a synthetic, self-assembling, virus-like particle platform that enables highly efficient vaccination., Self-assembling virus-like particles represent highly attractive tools for developing next-generation vaccines and protein therapeutics. We created ADDomer, an adenovirus-derived multimeric protein-based self-assembling nanoparticle scaffold engineered to facilitate plug-and-play display of multiple immunogenic epitopes from pathogens. We used cryo–electron microscopy at near-atomic resolution and implemented novel, cost-effective, high-performance cloud computing to reveal architectural features in unprecedented detail. We analyzed ADDomer interaction with components of the immune system and developed a promising first-in-kind ADDomer-based vaccine candidate to combat emerging Chikungunya infectious disease, exemplifying the potential of our approach.

Published
2019

3. New insights into HCV replication in original cells from Aedes mosquitoes

Author

Yassine Rechoum, Emmanuel Drouet, Frederic Garzoni, Pascal Fender, Olivier François, Alban Caporossi, Sylvie Larrat, Catherine Fallecker, Patrice Morand, Marie Anne Petit, Imre Berger, Institut de biologie structurale (IBS - UMR 5075 ), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Institut de Biologie et Pathologie [CHU Grenoble] (IBP), CHU Grenoble-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Biologie Computationnelle et Mathématique (TIMC-IMAG-BCM), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory (EMBL Grenoble), European Molecular Biology Laboratory [Grenoble] (EMBL), The School of Biochemistry [Bristol, U.K.], University of Bristol [Bristol], Centre de Recherche en Cancérologie de Lyon (UNICANCER/CRCL), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), C. F. is the recipient of MENRT fellowship (French Ministry of Higher Education and Research). I.B. and F.G. acknowledge funding from the European Commission (EC) Framework Program (FP) 7 through the projects ComplexINC (contract nr. 279,039) and Biostruct-X (contract nr. 283,570). This work used the platforms of the Grenoble Instruct Center (ISBG: UMS 3518 CNRS-CEA-UJF-EMBL) with support from FRISBI (ANR-10-INSB-05-02) and GRAL (ANR-10-LABX-49-01) within the Grenoble Partnership for Structural Biology (PSB)., ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), European Project: 279039,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,COMPLEXINC(2011), European Project: 283570,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2011-1,BIOSTRUCT-X(2011), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de biologie structurale ( IBS - UMR 5075 ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Institut de Biologie et Pathologie [CHU Grenoble] ( IBP ), CHU Grenoble-Université Grenoble Alpes ( UGA ), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications [Grenoble] ( TIMC-IMAG ), Université Joseph Fourier - Grenoble 1 ( UJF ) -Institut polytechnique de Grenoble - Grenoble Institute of Technology ( Grenoble INP ) -IMAG-Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ), Laboratoire européen de biologie moléculaire - European Molecular Biology Laboratory ( EMBL Grenoble ), European Molecular Biology Laboratory [Grenoble] ( EMBL ), University of Bristol [UK], Centre de Recherche en Cancérologie de Lyon ( CRCL ), Centre Léon Bérard [Lyon]-Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), ANR-10-INSB-05-02,FRISBI, ANR-10-LABX-49-01,Labex GRAL, European Project : 279039,EC:FP7:HEALTH,FP7-HEALTH-2011-two-stage,COMPLEXINC ( 2011 ), European Project : 283570,EC:FP7:INFRA,FP7-INFRASTRUCTURES-2011-1,BIOSTRUCT-X ( 2011 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-CHU Grenoble, BMC, BMC, Infrastructure Française pour la Biologie Structurale Intégrée - - FRISBI2010 - ANR-10-INBS-0005 - INBS - VALID, Grenoble Alliance for Integrated Structural Cell Biology - - GRAL2010 - ANR-10-LABX-0049 - LABX - VALID, New Technologies and Production Tools for Complex Protein Biologics - COMPLEXINC - - EC:FP7:HEALTH2011-11-01 - 2015-10-31 - 279039 - VALID, and Transnational access and enhancement of integrated Biological Structure determination at synchrotron X-ray radiation facilities - BIOSTRUCT-X - - EC:FP7:INFRA2011-09-01 - 2016-02-29 - 283570 - VALID

Subjects
0301 basic medicine, Human hepatocytes, [SDV]Life Sciences [q-bio], Hepacivirus, Virus Replication, Polymerase Chain Reaction, 0302 clinical medicine, Aedes aegypti, Viral Envelope Proteins, Aedes, Genotype, Phylogeny, Mosquito cell, chemistry.chemical_classification, education.field_of_study, biology, virus diseases, Hepatitis C virus (HCV), Aedes albopictus, Hepatitis C, 3. Good health, [SDV] Life Sciences [q-bio], Infectious Diseases, RNA, Viral, 030211 gastroenterology & hepatology, Sequence Analysis, Population, Cell Line, Microbiology, 03 medical and health sciences, Virology, Animals, Humans, education, Polymorphism, Genetic, Insect cell, [ SDV ] Life Sciences [q-bio], Research, fungi, biology.organism_classification, Insect Vectors, 030104 developmental biology, chemistry, Cell culture, Mutation, Hepatocytes, Peptides, Glycoprotein, HCV pseudo particles
Abstract

Background The existing literature about HCV association with, and replication in mosquitoes is extremely poor. To fill this gap, we performed cellular investigations aimed at exploring (i) the capacity of HCV E1E2 glycoproteins to bind on Aedes mosquito cells and (ii) the ability of HCV serum particles (HCVsp) to replicate in these cell lines. Methods First, we used purified E1E2 expressing baculovirus-derived HCV pseudo particles (bacHCVpp) so we could investigate their association with mosquito cell lines from Aedes aegypti (Aag-2) and Aedes albopictus (C6/36). We initiated a series of infections of both mosquito cells (Ae aegypti and Ae albopictus) with the HCVsp (Lat strain - genotype 3) and we observed the evolution dynamics of viral populations within cells over the course of infection via next-generation sequencing (NGS) experiments. Results Our binding assays revealed bacHCVpp an association with the mosquito cells, at comparable levels obtained with human hepatocytes (HepaRG cells) used as a control. In our infection experiments, the HCV RNA (+) were detectable by RT-PCR in the cells between 21 and 28 days post-infection (p.i.). In human hepatocytes HepaRG and Ae aegypti insect cells, NGS experiments revealed an increase of global viral diversity with a selection for a quasi-species, suggesting a structuration of the population with elimination of deleterious mutations. The evolutionary pattern in Ae albopictus insect cells is different (stability of viral diversity and polymorphism). Conclusions These results demonstrate for the first time that natural HCV could really replicate within Aedes mosquitoes, a discovery which may have major consequences for public health as well as in vaccine development. Electronic supplementary material The online version of this article (doi:10.1186/s12985-017-0828-z) contains supplementary material, which is available to authorized users.

Published
2016

4. Polyproteins in structural biology

Author

Maxime Chaillet, Frederic Garzoni, Thibaut Crépin, Alexandre Monod, Christopher Swale, Imre Berger, Thomas, Frank, Unit for Virus Host-Cell Interactions [Grenoble] (UVHCI), Université Joseph Fourier - Grenoble 1 (UJF)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Centre National de la Recherche Scientifique (CNRS), European Molecular Biology Laboratory [Grenoble] (EMBL), Unit of Virus Host Cell Interactions (UVHCI), and Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Protein Folding, Polyproteins, Protein Conformation, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein subunit, viruses, Molecular Sequence Data, 02 engineering and technology, Crystallography, X-Ray, Microscopy, Atomic Force, Article, 03 medical and health sciences, Synthetic biology, Viral Proteins, Protein structure, Structural Biology, Animals, Humans, Amino Acid Sequence, Molecular Biology, Polymerase, 030304 developmental biology, ComputingMethodologies_COMPUTERGRAPHICS, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], 021001 nanoscience & nanotechnology, Recombinant Proteins, 3. Good health, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Structural biology, Biochemistry, Proteome, Viruses, biology.protein, Protein folding, 0210 nano-technology
Abstract

Graphical abstract, Highlights • Structures have been determined for natural and recombinant polyproteins. • Native HIV Gag polyprotein architecture was revealed by cryo-EM of immature capsids. • Recombinant polyprotein technology has resolved sample preparation bottlenecks. • The high-resolution structure of influenza polymerase has been solved. • Single-molecule analysis of polyproteins revealed their folding characteristics., Polyproteins are chains of covalently conjoined smaller proteins that occur in nature as versatile means to organize the proteome of viruses including HIV. During maturation, viral polyproteins are typically cleaved into the constituent proteins with different biological functions by highly specific proteases, and structural analyses at defined stages of this maturation process can provide clues for antiviral intervention strategies. Recombinant polyproteins that use similar mechanisms are emerging as powerful tools for producing hitherto inaccessible protein targets such as the influenza polymerase, for high-resolution structure determination by X-ray crystallography. Conversely, covalent linking of individual protein subunits into single polypeptide chains are exploited to overcome sample preparation bottlenecks. Moreover, synthetic polyproteins provide a promising tool to dissect dynamic folding of polypeptide chains into three-dimensional architectures in single-molecule structure analysis by atomic force microscopy (AFM). The recent use of natural and synthetic polyproteins in structural biology and major achievements are highlighted in this contribution.

Published
2015

5. The architecture of human general transcription factor TFIID core complex

Author

Frederic Garzoni, Petros Papadopoulos, Imre Berger, Christoph Bieniossek, Maxime Chaillet, Elisabeth Scheer, Laszlo Tora, Gabor Papai, Patrick Schultz, Christiane Schaffitzel, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), and Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, TATA box, [SDV]Life Sciences [q-bio], genetic processes, information science, macromolecular substances, Biology, 03 medical and health sciences, 0302 clinical medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Cells, Cultured, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Genetics, 0303 health sciences, Multidisciplinary, General transcription factor, fungi, Cryoelectron Microscopy, Cell biology, Protein Structure, Tertiary, TAF1, TAF4, TAF2, Transcription Factor TFIID, health occupations, Transcription factor II D, 030217 neurology & neurosurgery, Transcription factor II A, HeLa Cells, Protein Binding
Abstract

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 A resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8-TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.

Published
2013

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