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7 results on '"Pauline Macheboeuf"'

1. MagC is a NplC/P60‐like member of the $\alpha$‐2‐macroglobulin Mag complex of $Pseudomonas\ aeruginosa$ that interacts with peptidoglycan

Author

Ina Attrée, Andréa Dessen, Daniel M. Trindade, Carlos Contreras-Martel, Pauline Macheboeuf, Samira Zouhir, Brazilian Biosciences National Laboratory (LNBio), National Center for Research in Energy and Materials, 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), Pathogenèse bactérienne et réponses cellulaires (PBRC), Centre National de la Recherche Scientifique (CNRS)-Biologie du Cancer et de l'Infection (BCI ), 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)-Institut National de la Santé et de la Recherche Médicale (INSERM)-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)-Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-10-INBS-0005,FRISBI,Infrastructure Française pour la Biologie Structurale Intégrée(2010), ANR-17-EURE-0003,CBH-EUR-GS,CBH-EUR-GS(2017), Groupe Pathogenèse Bactérienne et Réponses Cellulaires / Bacterial Pathogenesis and Cellular Responses Group (IBS-PBRC), 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)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG)

Subjects
Operon, medicine.medical_treatment, [SDV]Life Sciences [q-bio], Biophysics, Peptidoglycan, Calorimetry, medicine.disease_cause, Crystallography, X-Ray, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, Structural Biology, Genetics, medicine, Amino Acid Sequence, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Protease, Sequence Homology, Amino Acid, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Pseudomonas aeruginosa, 030302 biochemistry & molecular biology, Isothermal titration calorimetry, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Cell Biology, Pregnancy-Associated alpha 2-Macroglobulins, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Macroglobulin, chemistry, Macroglobulins, Ultracentrifugation, Function (biology), Protein Binding
Abstract

International audience; Bacterial $\alpha$-2 macroglobulins (A2Ms) structurally resemble the large spectrum protease inhibitors of the eukaryotic immune system. In $Pseudomonas\ aeruginosa$, MagD acts as an A2M and is expressed within a six-gene operon encoding the MagA-F proteins. In this work, we employ isothermal calorimetry (ITC), analytical ultracentrifugation (AUC), and X-ray crystallography to investigate the function of MagC and show that MagC associates with the macroglobulin complex and with the peptidoglycan (PG). However, the catalytic residues of MagC display an inactive conformation that could suggest that it binds to PG but does not degrade it. We hypothesize that MagC could serve as an anchor between the MagD macroglobulin and the PG and could provide stabilization and/or regulation for the entire complex.

Published
2021
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2. Human viperin catalyzes the modification of GPP and FPP potentially affecting cholesterol synthesis

Author

Patricia Amara, Elena A. Andreeva, Winfried Weissenhorn, Pauline Macheboeuf, Yvain Nicolet, Pavel Mikulecky, 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), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, Models, Molecular, Oxidoreductases Acting on CH-CH Group Donors, Biophysics, Farnesyl pyrophosphate, Biochemistry, Substrate Specificity, 03 medical and health sciences, chemistry.chemical_compound, Polyisoprenyl Phosphates, Structural Biology, Viperin, Genetics, Humans, radical SAM, Molecular Biology, Lipid raft, innate immunity, Virus Release, chemistry.chemical_classification, 030102 biochemistry & molecular biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Cholesterol, Geranyl pyrophosphate, Proteins, Cell Biology, Molecular Docking Simulation, 030104 developmental biology, Enzyme, chemistry, Biocatalysis, antiviral activity, lipids (amino acids, peptides, and proteins), Mevalonate pathway, Radical SAM, Sesquiterpenes
Abstract

International audience; Viperin is a radical SAM enzyme that possesses antiviral properties against a broad range of enveloped viruses. Here we describe the activity of human viperin with two molecules of the mevalonate pathway, geranyl pyrophosphate and farnesyl pyrophosphate, involved in cholesterol biosynthesis. We postulate that the radical modification of these two molecules by viperin might lead to defects in cholesterol synthesis, thereby affecting the composition of lipid rafts and subsequent enveloped virus budding. This article is protected by copyright. All rights reserved.

Published
2018
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3. Intrinsic Dynamics in ECFP and Cerulean Control Fluorescence Quantum Yield

Author

Richard Chazal, Christelle Lazareno-Saez, Marjolaine Noirclerc-Savoye, Mickaël Lelimousin, Pauline Macheboeuf, Martin J. Field, Bernhard Paetzold, Antoine Royant, Sophie Le Vot, and Dominique Bourgeois

Subjects
Models, Molecular, Quenching (fluorescence), Protein Conformation, Chemistry, Cerulean, Green Fluorescent Proteins, Tryptophan, Hydrogen-Ion Concentration, Chromophore, Crystallography, X-Ray, Photochemistry, Biochemistry, Fluorescence, Recombinant Proteins, Molecular dynamics, Protein structure, Förster resonance energy transfer, Fluorescence Resonance Energy Transfer, Biophysics, Computer Simulation, Denaturation (biochemistry), Fluorescent Dyes
Abstract

Enhanced cyan fluorescent protein (ECFP) and its variant Cerulean are genetically encoded fluorophores widely used as donors in FRET-based cell imaging experiments. First, we have confirmed through denaturation experiments that the double-peak spectroscopic signature of these fluorescent proteins originates from the indole ring of the chromophore. Then, to explain the improvement in the fluorescence properties of Cerulean compared to those of ECFP, we have determined the high-resolution crystal structures of these two proteins at physiological pH and performed molecular dynamics simulations. In both proteins, the N-terminal half of the seventh strand exhibits two conformations. These conformations both have a complex set of van der Waals interactions with the chromophore and, as our simulations suggest, they interconvert on a nanosecond time scale. The Y145A and H148D mutations in Cerulean stabilize these interactions and allow the chromophore to be more planar, better packed, and less prone to collisional quenching, albeit only intermittently. As a consequence, the probability of nonradiative decay is significantly decreased. Our results highlight the considerable dynamical flexibility that exists in the vicinity of the tryptophan-based chromophore of these engineered fluorescent proteins and provide insights that should allow the design of mutants with enhanced optical properties.

Published
2009
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4. Asymmetric ring structure of Vps4 required for ESCRT-III disassembly

Author

Elisabetta Boeri-Erba, Yuanfei Wu, Grégory Effantin, Pauline Macheboeuf, Heinrich G. Göttlinger, Winfried Weissenhorn, Patricia Renesto, Christophe Caillat, Andrew A. McCarthy, Institut de biologie structurale [1992-2019] (IBS - UMR 5075 [1992-2019]), 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), Program in Gene Function and Expression, Program in Molecular Medicine, University of Massachusetts Medical School [Worcester] (UMASS), University of Massachusetts System (UMASS)-University of Massachusetts System (UMASS), Thérapeutique Recombinante Expérimentale [?-2015] (TIMC-TheREx [?-2015]), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 [2011-2015] (TIMC [2011-2015]), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-IMAG-Institut polytechnique de Grenoble - Grenoble Institute of Technology [2007-2019] (Grenoble INP [2007-2019])-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-IMAG-Institut polytechnique de Grenoble - Grenoble Institute of Technology [2007-2019] (Grenoble INP [2007-2019])-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS), Institut de biologie structurale (IBS - UMR 5075 ), European Molecular Biology Laboratory [Grenoble] (EMBL), Thérapeutique Recombinante Expérimentale (TIMC-IMAG-TheREx), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Université Joseph Fourier - Grenoble 1 (UJF)-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 Joseph Fourier - Grenoble 1 (UJF)-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 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), and VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)

Subjects
Models, Molecular, Protein Conformation, General Physics and Astronomy, Protomer, macromolecular substances, Biology, General Biochemistry, Genetics and Molecular Biology, ESCRT, Article, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Protein structure, Adenosine Triphosphate, ATP hydrolysis, Binding site, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Vacuolar protein sorting, 0303 health sciences, Multidisciplinary, Binding Sites, Endosomal Sorting Complexes Required for Transport, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], General Chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], chemistry, Biochemistry, Sulfolobaceae, Mutation, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Biophysics, HIV-1, ADP binding, Gene Expression Regulation, Archaeal, Adenosine triphosphate, 030217 neurology & neurosurgery
Abstract

The vacuolar protein sorting 4 AAA–ATPase (Vps4) recycles endosomal sorting complexes required for transport (ESCRT-III) polymers from cellular membranes. Here we present a 3.6-Å X-ray structure of ring-shaped Vps4 from Metallosphera sedula (MsVps4), seen as an asymmetric pseudohexamer. Conserved key interface residues are shown to be important for MsVps4 assembly, ATPase activity in vitro, ESCRT-III disassembly in vitro and HIV-1 budding. ADP binding leads to conformational changes within the protomer, which might propagate within the ring structure. All ATP-binding sites are accessible and the pseudohexamer binds six ATP with micromolar affinity in vitro. In contrast, ADP occupies one high-affinity and five low-affinity binding sites in vitro, consistent with conformational asymmetry induced on ATP hydrolysis. The structure represents a snapshot of an assembled Vps4 conformation and provides insight into the molecular motions the ring structure undergoes in a concerted action to couple ATP hydrolysis to ESCRT-III substrate disassembly., Vps4 is a AAA+ family protein involved in the disassembly of ESCRT-III polymers during membrane fission events such as occur during HIV budding. Here the authors propose a structure-based model of how the conformational flexibility of Vps4 can be translated into mechanical forces to disassemble ESCRT-III during membrane fission.

Published
2015
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5. Structural basis for ESCRT-III CHMP3 recruitment of AMSH

Author

David Lutje Hulsik, Bettina Hartlieb, Pauline Macheboeuf, Yoshiko Usami, Charles Sabin, Winfried Weissenhorn, Julianna Solomons, Heinrich G. Göttlinger, Emilie Poudevigne, 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), 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), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), 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), and Thomas, Frank

Subjects
MESH: Protein Structure, Quaternary, MESH: Protein Structure, Secondary, HIV Infections, MESH: Amino Acid Sequence, Protein Structure, Secondary, Deubiquitinating enzyme, MESH: Endosomal Sorting Complexes Required for Transport, MESH: HIV-1, MESH: Protein Structure, Tertiary, 0302 clinical medicine, Protein structure, Ubiquitin, Structural Biology, MESH: Peptide Fragments, Virus Release, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Virus Release, MESH: HIV Infections, 3. Good health, Biochemistry, MESH: HEK293 Cells, Ubiquitin Thiolesterase, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Endosome, Molecular Sequence Data, macromolecular substances, ESCRT, Article, 03 medical and health sciences, Humans, Amino Acid Sequence, MESH: Hydrogen Bonding, Binding site, Protein Structure, Quaternary, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Molecular Sequence Data, Endosomal Sorting Complexes Required for Transport, Hydrogen Bonding, MESH: Ubiquitin Thiolesterase, MESH: Multiprotein Complexes, Peptide Fragments, Protein Structure, Tertiary, HEK293 Cells, Multiprotein Complexes, biology.protein, Biophysics, HIV-1, 030217 neurology & neurosurgery, Cytokinesis
Abstract

International audience; Endosomal sorting complexes required for transport (ESCRT) recognize ubiquitinated cargo and catalyze diverse budding processes including multivesicular body biogenesis, enveloped virus egress, and cytokinesis. We present the crystal structure of an N-terminal fragment of the deubiquitinating enzyme AMSH (AMSHΔC) in complex with the C-terminal region of ESCRT-III CHMP3 (CHMP3ΔN). AMSHΔC folds into an elongated 90 Å long helical assembly that includes an unusual MIT domain. CHMP3ΔN is unstructured in solution and helical in complex with AMSHΔC, revealing a novel MIT domain interacting motif (MIM) that does not overlap with the CHMP1-AMSH binding site. ITC and SPR measurements demonstrate an unusual high-affinity MIM-MIT interaction. Structural analysis suggests a regulatory role for the N-terminal helical segment of AMSHΔC and its destabilization leads to a loss of function during HIV-1 budding. Our results indicate a tight coupling of ESCRT-III CHMP3 and AMSH functions and provide insight into the regulation of ESCRT-III.

Published
2011

6. Solution X-ray scattering study of a full-length class A penicillin-binding protein

Author

M. Piuzzi, S. Finet, Andréa Dessen, P. Vachette, F. Bontems, J. Pérez, Pauline Macheboeuf, Institut de Chimie des Substances Naturelles (ICSN), and Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)

Subjects
Penicillin binding proteins, Biophysics, Biochemistry, Bacterial cell structure, 03 medical and health sciences, chemistry.chemical_compound, Glycosyltransferase, Scattering, Small Angle, polycyclic compounds, Molecule, Penicillin-Binding Proteins, Homology modeling, Molecular Biology, 030304 developmental biology, 0303 health sciences, biology, Small-angle X-ray scattering, [CHIM.ORGA]Chemical Sciences/Organic chemistry, X-Rays, 030302 biochemistry & molecular biology, Cell Biology, Protein Structure, Tertiary, Crystallography, Monomer, Streptococcus pneumoniae, chemistry, Models, Chemical, biology.protein, Peptidoglycan
Abstract

International audience; Penicillin binding proteins (PBPs) catalyze essential steps in the biosynthesis of peptidoglycan, the main component of the bacterial cell wall. PBPs can harbor two catalytic domains, namely the glycosyltransferase (GT) and transpeptidase (TP) activities, the latter being the target for β-lactam antibiotics. Despite the availability of structural information regarding bi-functional PBPs, little is known regarding the interaction and flexibility between the TP and GT domains. Here, we describe the structural characterization in solution by small angle X-ray scattering (SAXS) of PBP1b, a bi-functional PBP from Streptococcus pneumoniae. The molecule is present in solution as an elongated monomer. Refinement of internal coordinates starting from a homology model yields models in which the two domains are in an extended conformation without any mutual contact compatible with the existence of restricted mobility.

Published
2011
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7. A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

Author

Laura E. McCoy, Simone Battella, Charles Sabin, Alexandre M. J. J. Bonvin, Andreas Hinz, Robin A. Weiss, Michael S. Seaman, David Lutje Hulsik, Lucy Rutten, Miriam Hock, C. Theo Verrips, Alejandra Ramos, Winfried Weissenhorn, Nika M. Strokappe, Pauline Macheboeuf, Johannes P. M. Langedijk, Mohamed El Khattabi, Adrien Favier, Pascal Poignard, Ying-ying Liu, David Davis, Marlén M. I. Aasa-Chapman, Jean-Pierre Simorre, Anna Forsman Quigley, 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), Departments of Applied Physics [New Haven], Yale University [New Haven], Research Department of Infection and Population Health [London], University College of London [London] (UCL), Independent Department for Medical Psychology and Medical Sociology, Universität Leipzig, Beth Israel Deaconess Medical Center [Boston] (BIDMC), Harvard Medical School [Boston] (HMS), Centre d'Immunologie de Marseille - Luminy (CIML), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), 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), ANR-10-LABX-0049,GRAL,Grenoble Alliance for Integrated Structural Cell Biology(2010), Centre National de la Recherche Scientifique (CNRS)-European Molecular Biology Laboratory [Grenoble] (EMBL)-Université Joseph Fourier - Grenoble 1 (UJF), Universität Leipzig [Leipzig], Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and 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)

Subjects
Macromolecular Assemblies, MESH: HIV Envelope Protein gp41, MESH: Amino Acid Sequence, Complementarity determining region, Adaptive Immunity, HIV Antibodies, MESH: Base Sequence, Antibody production, Neutralization, Epitope, MESH: Antibodies, Neutralizing, Epitopes, Immunodeficiency Viruses, Bacteriophages, MESH: Animals, Biomacromolecule-Ligand Interactions, Enzyme-linked immunoassays, lcsh:QH301-705.5, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Hydrophobic and Hydrophilic Interactions, MESH: Neutralization Tests, 030302 biochemistry & molecular biology, Antivirals, Immunizations, Lipids, HIV Envelope Protein gp41, MESH: Surface Plasmon Resonance, 3. Good health, MESH: Mutagenesis, Site-Directed, International (English), Viral Envelope, MESH: Camelids, New World, MESH: Complementarity Determining Regions, MESH: Immunization, Antibody, Camelids, New World, Hydrophobic and Hydrophilic Interactions, Research Article, MESH: Proteolipids, lcsh:Immunologic diseases. Allergy, MESH: Epitopes, medicine.drug_class, Proteolipids, Molecular Sequence Data, Immunology, Biophysics, Membrane fusion, Viral Structure, Biology, Monoclonal antibody, Gp41, MESH: Single-Domain Antibodies, Microbiology, Antibodies, Cell Line, 03 medical and health sciences, Viral envelope, Neutralization Tests, ddc:570, Virology, Genetics, medicine, Animals, Humans, Amino Acid Sequence, Microbial Pathogens, Molecular Biology, 030304 developmental biology, MESH: Humans, MESH: Molecular Sequence Data, Base Sequence, MESH: HIV Antibodies, Crystal structure, Immunity, Viral Vaccines, Single-Domain Antibodies, Surface Plasmon Resonance, Antibodies, Neutralizing, Complementarity Determining Regions, Molecular biology, MESH: Cell Line, lcsh:Biology (General), chemistry, Mutagenesis, Site-Directed, HIV-1, biology.protein, Immunization, Parasitology, lcsh:RC581-607, Glycoprotein
Abstract

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10., Author Summary Due to the absence of an effective vaccine or cure for acquired immunodeficiency syndrome (AIDS), HIV-1 infections still result in high mortality. Two antibodies, 2F5 and 4E10, previously isolated from HIV-1 infected patients, prevent infections by binding to the MPER of gp41, a part of the virus that is difficult to access and only transiently exposed. Here, we immunized llamas with a gp41-based immunogen and subsequently isolated a small antibody fragment (VHH) that can easily access and recognize the MPER. We showed that a unit of two VHH, named bi-2H10, was indeed capable of preventing HIV-1 from infecting cells. We determined the three dimensional structure of the VHH and mapped its interaction site to an MPER region that overlaps with the 2F5 epitope. The 2H10 VHH displays a membrane binding component important for neutralization that resembles that of 2F5. In conclusion, we have developed an immunogen and a small antibody that may have great potential for development of novel anti-HIV/AIDS vaccines and treatments.

Published
2013
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