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1. Structural basis for haem piracy from host haemopexin by Haemophilus influenzae

Author

Mohamed Chami, Silvia Zambolin, Sylviane Hoos, Ahmed Haouz, Philippe Delepelaire, Vincent Villeret, Bernard Clantin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 (UGSF), Université de Lille-Centre National de la Recherche Scientifique (CNRS), Center for Cellular Imaging and Nanoanalytics, University of Basel (Unibas), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Cristallographie (Plateforme) - Crystallography (Platform), This work was supported by grants from ANR (HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 and LABEX DYNAMO ANR-11-LABEX-0011-01)., ANR-12-BSV3-0022,HEMESTOCKEXCHANGE,Hème et porphyrine chez des bactéries modèle: des fonctions aux mécanismes(2012), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Université de Lille-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), CNRS, Université de Lille, Laboratoire de biologie physico-chimique des protéines membranaires [LBPC-PM (UMR_7099)], and Unité de Glycobiologie Structurale et Fonctionnelle UMR 8576 [UGSF]

Subjects
0301 basic medicine, Receptors, Peptide, Science, General Physics and Astronomy, Plasma protein binding, Heme, Biology, blood proteins, medicine.disease_cause, General Biochemistry, Genetics and Molecular Biology, Article, Haemophilus influenzae, 03 medical and health sciences, Bacterial Proteins, Microscopy, Electron, Transmission, X-Ray Diffraction, Haem transport, Heme metabolism, medicine, polycyclic compounds, Escherichia coli, Animals, Multidisciplinary, Molecular Structure, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], digestive, oral, and skin physiology, General Chemistry, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, 3. Good health, 030104 developmental biology, Biochemistry, Rabbits
Abstract

Haemophilus influenzae is an obligate human commensal/pathogen that requires haem for survival and can acquire it from several host haemoproteins, including haemopexin. The haem transport system from haem-haemopexin consists of HxuC, a haem receptor, and the two-partner-secretion system HxuB/HxuA. HxuA, which is exposed at the cell surface, is strictly required for haem acquisition from haemopexin. HxuA forms complexes with haem-haemopexin, leading to haem release and its capture by HxuC. The key question is how HxuA liberates haem from haemopexin. Here, we solve crystal structures of HxuA alone, and HxuA in complex with the N-terminal domain of haemopexin. A rational basis for the release of haem from haem-haemopexin is derived from both in vivo and in vitro studies. HxuA acts as a wedge that destabilizes the two-domains structure of haemopexin with a mobile loop on HxuA that favours haem ejection by redirecting key residues in the haem-binding pocket of haemopexin., Haemophilus influenzae requires haem, and acquires it from host haemoproteins including haemopexin. Here, the authors examine the haem transport system consisting of HxuA, HxuB and HxuC via the structures of HxuA in complex with haemopexin.

Published
2016
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2. The Stable Interaction Between Signal Peptidase LepB of Escherichia coli and Nuclease Bacteriocins Promotes Toxin Entry into the Cytoplasm

Author

Miklos de Zamaroczy, Jacques Oberto, Karine Moncoq, Liliana Mora, Patrick England, Expression Génétique Microbienne (EGM (UMR_8261 / FRE_3630)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Biophysique des macromolécules et leurs interactions, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Biologie Cellulaire des Archées (ARCHEE), Département Microbiologie (Dpt Microbio), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), This work was supported by the CNRS (France)., Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Institut de Biologie Intégrative de la Cellule (I2BC), Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay-Université Paris-Sud - Paris 11 (UP11)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Saclay, Expression Génétique Microbienne ( EGM ), Université Paris Diderot - Paris 7 ( UPD7 ) -Institut de Biologie Physico-Chimique-Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de biologie physico-chimique des protéines membranaires ( LBPC-PM ), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Institut de Biologie Intégrative de la Cellule ( I2BC ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ), Biologie Cellulaire des Archées ( ARCHEE ), Département Microbiologie ( Dpt Microbio ), Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Institut de Biologie Intégrative de la Cellule ( I2BC ), and Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Université Paris-Saclay-Centre National de la Recherche Scientifique ( CNRS )

Subjects
Cytoplasm, LepB, Amino Acid Motifs, MESH: Escherichia coli Proteins, MESH: Amino Acid Sequence, Biochemistry, endonuclease, MESH: Amino Acid Motifs, MESH: Protein Structure, Tertiary, MESH: Bacteriocins, Bacteriocins, MESH: Serine Endopeptidases, Peptide sequence, membrane, 0303 health sciences, Signal peptidase, Deoxyribonucleases, biology, MESH: Escherichia coli, Escherichia coli Proteins, Serine Endopeptidases, Colicin, MESH: Membrane Proteins, colicin, Protein Binding, MESH: Ribonucleases, MESH: Biological Transport, Bacterial Toxins, Molecular Sequence Data, MESH: Sequence Alignment, Sequence alignment, 03 medical and health sciences, Ribonucleases, Bacteriocin, bacteriocin, Membrane Biology, [SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN], Escherichia coli, Inner membrane, MESH: Protein Binding, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Amino Acid Sequence, Molecular Biology, 030304 developmental biology, Nuclease, MESH: Molecular Sequence Data, [ SDV ] Life Sciences [q-bio], 030306 microbiology, MESH: Cytoplasm, microbiology, Membrane Proteins, toxicity, Biological Transport, protease, Cell Biology, Periplasmic space, biochemical phenomena, metabolism, and nutrition, Protein Structure, Tertiary, MESH: Bacterial Toxins, biology.protein, bacteria, protein import, Sequence Alignment, MESH: Deoxyribonucleases
Abstract

International audience; LepB is a key membrane component of the cellular secretion machinery, which releases secreted proteins into the periplasm by cleaving the inner membrane-bound leader. We showed that LepB is also an essential component of the machinery hijacked by the tRNase colicin D for its import. Here we demonstrate that this non-catalytic activity of LepB is to promote the association of the central domain of colicin D with the inner membrane before the FtsH-dependent proteolytic processing and translocation of the toxic tRNase domain into the cytoplasm. The novel structural role of LepB results in a stable interaction with colicin D, with a stoichiometry of 1:1 and a nanomolar Kd determined in vitro. LepB provides a chaperone-like function for the penetration of several nuclease-type bacteriocins into target cells. The colicin-LepB interaction is shown to require only a short peptide sequence within the central domain of these bacteriocins and to involve residues present in the short C-terminal Box E of LepB. Genomic screening identified the conserved LepB binding motif in colicin-like ORFs from 13 additional bacterial species. These findings establish a new paradigm for the functional adaptability of an essential inner-membrane enzyme.

Published
2015
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3. Effect of amphiphilic environment on the solution structure of mouse TSPO translocator protein

Author

Sophie Combet, Françoise Bonneté, Stéphanie Finet, Alexandre Pozza, Christelle Saade, Anne Martel, Alexandros Koutsioubas, Jean-Jacques Lacapère, Laboratoire Léon Brillouin (LLB - UMR 12), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), LLB - Matière molle et biophysique (MMB), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut de minéralogie, de physique des matériaux et de cosmochimie (IMPMC), Muséum national d'Histoire naturelle (MNHN)-Institut de recherche pour le développement [IRD] : UR206-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Mécanique et d'Acoustique [Marseille] (LMA ), Aix Marseille Université (AMU)-École Centrale de Marseille (ECM)-Centre National de la Recherche Scientifique (CNRS), Institut Laue-Langevin (ILL), Jülich Centre for Neutron Science (JCNS), Forschungszentrum Jülich GmbH at Heinz Maier-Leibnitz Zentrum (MLZ), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), and CNRS (MITI 2021 and Tremplin@ INP 2021)

Subjects
[SDV]Life Sciences [q-bio], ddc:540, General Medicine, Biochemistry
Abstract

International audience; The translocator protein (TSPO) is a ubiquitous transmembrane protein of great pharmacological interest thanks to its high affinity to many drug ligands. The only high-resolution 3D-structure known for mammalian TSPO was obtained by NMR for the mouse mTSPO in DPC detergent only in presence of the high-affinity PK 11195 ligand. An atomic structure of free-ligand mTSPO is still missing to better understand the interaction of ligands with mTSPO and their effects on the protein conformation.Here, we decipher the solution structures of the recombinant mTSPO without ligand both in (i) SDS, the detergent used to extract and purify the protein from E. coli inclusion bodies, and (ii) DPC, the detergent used to solve the PK 11195-binding mTSPO NMR structure.We report partially refolded and less flexible mTSPO helices in DPC compared to SDS. Besides, DPC stabilizes the tertiary structure of mTSPO, as shown by a higher intrinsic Trp fluorescence and changes in indole environment.We evaluate by SEC-MALLS that ∼135 SDS and ∼100 DPC molecules are bound to mTSPO. SEC-small-angle X-ray (SAXS) and neutron (SANS) scattering confirm a larger mTSPO-detergent complex in SDS than in DPC. Using the contrast-matching technique in SEC-SANS, we demonstrate that mTSPO conformation is more compact and less flexible in DPC than in SDS. Combining ab initio modeling with SANS, we confirm that mTSPO conformation is less elongated in DPC than in SDS. However, the free-ligand mTSPO envelope in DPC is not as compact as the PK 11195-binding protein NMR structure, the ligand stiffening the protein.

Published
2023
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4. Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids

Author

Anaïs Marconnet, Baptiste Michon, Bastien Prost, Audrey Solgadi, Christel Le Bon, Fabrice Giusti, Christophe Tribet, Manuela Zoonens, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Ingénierie et Plateformes au Service de l'Innovation Thérapeutique (IPSIT), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and zoonens, manuela

Subjects
Polymers, [SDV]Life Sciences [q-bio], Detergents, Maleates, membrane proteins, Cycloparaffins, amphipols, Lipids, surfactants, Carbon, Analytical Chemistry, [SDV] Life Sciences [q-bio], SMA copolymers, Bacteriorhodopsins, extraction, Polystyrenes
Abstract

International audience; One of the biggest challenges in membrane protein (MP) research is to secure physiologically relevant structural and functional information after extracting MPs from their native membrane. Amphipathic polymers represent attractive alternatives to detergents for stabilizing MPs in aqueous solutions. The predominant polymers used in MP biochemistry and biophysics are amphipols (APols), one class of which, styrene maleic-acid (SMA) copolymers and their derivatives, has proven particularly efficient at MP extraction. In order to examine the relationship between the chemical structure of the polymers and their ability to extract MPs, we have developed two novel classes of APols bearing either cycloalkane or aryl (aromatic) rings, named CyclAPols and ArylAPols, respectively. The effect on solubilization of such parameters as the density of hydrophobic groups, the number of carbon atoms and their arrangement in the hydrophobic moieties, as well as the charge density of the polymers was evaluated. The membrane-solubilizing efficiency of the SMAs, CyclAPols and ArylAPols was compared using as models i) two MPs, BmrA and a GFP-fused version of LacY, overexpressed in the inner membrane of Escherichia coli, and ii) bacteriorhodopsin, naturally expressed in the purple membrane of Halobacterium salinarum. This analysis shows that, as compared to SMAs, the novel APols feature an improved efficiency at extracting MPs while preserving native protein-lipid interactions.

Published
2022
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5. Structural and molecular determinants for the interaction of ExbB from Serratia marcescens and HasB, a TonB paralog

Author

Valérie Biou, Ricardo Jorge Diogo Adaixo, Mohamed Chami, Pierre-Damien Coureux, Benoist Laurent, Véronique Yvette Ntsogo Enguéné, Gisele Cardoso de Amorim, Nadia Izadi-Pruneyre, Christian Malosse, Julia Chamot-Rooke, Henning Stahlberg, Philippe Delepelaire, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Center for Cellular Imaging and Nanoanalytics, University of Basel (Unibas), Laboratoire de Biologie Structurale de la Cellule (BIOC), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS), University of Cambridge [UK] (CAM), Bioinformatique structurale - Structural Bioinformatics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre de Bioinformatique, Biostatistique et Biologie Intégrative (C3BI), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Universidade Federal do Rio de Janeiro (UFRJ), Spectrométrie de Masse pour la Biologie – Mass Spectrometry for Biology (UTechS MSBio), Institut Pasteur [Paris] (IP)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was supported by grants from the ANR (HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 and LABEX DYNAMO ANR-11-LABEX-0011-01)., ANR-12-BSV3-0022,HEMESTOCKEXCHANGE,Hème et porphyrine chez des bactéries modèle: des fonctions aux mécanismes(2012), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Biou, Valérie [0000-0003-1600-6717], Coureux, Pierre-Damien [0000-0002-1328-7229], Enguéné, Véronique Yvette Ntsogo [0000-0003-0096-3192], de Amorim, Gisele Cardoso [0000-0003-4348-9515], Izadi-Pruneyre, Nadia [0000-0002-6864-2961], Chamot-Rooke, Julia [0000-0002-9427-543X], Delepelaire, Philippe [0000-0002-5190-7118], and Apollo - University of Cambridge Repository

Subjects
[SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, 101, Escherichia coli Proteins, 101/28, 101/6, article, Medicine (miscellaneous), MESH: Escherichia coli Proteins, Heme, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, General Biochemistry, Genetics and Molecular Biology, 631/45/535/1258/1259, 82/80, MESH: Serratia marcescens, MESH: Heme, Escherichia coli, MESH: Protein Binding, 631/326/41/2536, General Agricultural and Biological Sciences, 82/83, Serratia marcescens, Protein Binding
Abstract

Funder: LABEX DYNAMO ANR-11-LABEX-0011-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01, Funder: ANR HEMESTOCKEXCHANGE ANR-12-BSV3-0022-01 ANR LABEX DYNAMO ANR-11-LABEX-0011-01, ExbB and ExbD are cytoplasmic membrane proteins that associate with TonB to convey the energy of the proton-motive force to outer membrane receptors in Gram-negative bacteria for iron uptake. The opportunistic pathogen Serratia marcescens (Sm) possesses both TonB and a heme-specific TonB paralog, HasB. ExbBSm has a long periplasmic extension absent in other bacteria such as E. coli (Ec). Long ExbB’s are found in several genera of Alphaproteobacteria, most often in correlation with a hasB gene. We investigated specificity determinants of ExbBSm and HasB. We determined the cryo-EM structures of ExbBSm and of the ExbB-ExbDSm complex from S. marcescens. ExbBSm alone is a stable pentamer, and its complex includes two ExbD monomers. We showed that ExbBSm extension interacts with HasB and is involved in heme acquisition and we identified key residues in the membrane domain of ExbBSm and ExbBEc, essential for function and likely involved in the interaction with TonB/HasB. Our results shed light on the class of inner membrane energy machinery formed by ExbB, ExbD and HasB.

Published
2022
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6. Rab27A and its effector MyRIP link secretory granules to F-actin and control their motion towards release sites

Author

Isabelle Fanget, Sébastien Huet, François Darchen, Christine Petit, Catherine Chapuis, Graça Raposo, Viet Samuel Tran, Sophie Cribier, Geneviève de Saint Basile, Gaël Ménasché, Aziz El-Amraoui, Claire Desnos, Jean-Sébastien Schonn, Jean-Pierre Henry, Biologie cellulaire et moléculaire de la sécrétion (BCMS), Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC), Génétique des Déficits sensoriels, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Compartimentation et dynamique cellulaires (CDC), Centre National de la Recherche Scientifique (CNRS)-Institut Curie-Université Pierre et Marie Curie - Paris 6 (UPMC), Inserm U429 (CHU Necker - Enfants Malades [AP-HP]), Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Necker - Enfants Malades [AP-HP], Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), J.-S. Schonn was supported by a fellowship from the Fondation de la Recherche Médicale, and S. Huet was supported by the Direction Générale de l’Armement. This work was supported by a grant from the Ministère de la Recherche (DRAB)., Centre National de la Recherche Scientifique (CNRS)-Institut Curie [Paris]-Université Pierre et Marie Curie - Paris 6 (UPMC), Developpement Normal et Pathologique du Système Immunitaire, Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Chaire Génétique et physiologie cellulaire, Collège de France (CdF (institution)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), J.-S. Schonn was supported by a fellowship from the Fondation de la Recherche Médicale, and S. Huet was supported by the Direction Générale de l'Armement. This work was supported by a grant from the Ministère de la Recherche (DRAB)., Chapuis, Catherine, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Curie [Paris]-Centre National de la Recherche Scientifique (CNRS), Collège de France - Chaire Génétique et physiologie cellulaire, Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biologie cellulaire et moléculaire de la sécrétion ( BCMS ), Centre National de la Recherche Scientifique ( CNRS ), Institut de Génétique et Développement de Rennes ( IGDR ), Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -Centre National de la Recherche Scientifique ( CNRS ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique ( CNRS ), Compartimentation et dynamique cellulaires ( CDC ), Centre National de la Recherche Scientifique ( CNRS ) -INSTITUT CURIE-Université Pierre et Marie Curie - Paris 6 ( UPMC ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de biologie physico-chimique des protéines membranaires ( LBPC-PM ), and Université Paris Diderot - Paris 7 ( UPD7 ) -Institut de Biologie Physico-Chimique-Centre National de la Recherche Scientifique ( CNRS )

Subjects
Chromaffin Cells, [SDV]Life Sciences [q-bio], Rab27A, MyRIP, exocytosis, actin, neuroendocrine cell, [SDV.BC.BC]Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], PC12 Cells, rab27 GTP-Binding Proteins, 0302 clinical medicine, Depsipeptides, Myosin, 0303 health sciences, Secretory Vesicle, Cell biology, Actin Cytoskeleton, Melanophilin, Thiazolidines, endocrine system, Myosin Type V, macromolecular substances, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Peptides, Cyclic, Exocytosis, Article, 03 medical and health sciences, [SDV.BC.BC] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Animals, RAB27, Actin, 030304 developmental biology, Adaptor Proteins, Signal Transducing, Myosin Heavy Chains, Secretory Vesicles, [ SDV.BC.BC ] Life Sciences [q-bio]/Cellular Biology/Subcellular Processes [q-bio.SC], Cell Biology, Actin cytoskeleton, Bridged Bicyclo Compounds, Heterocyclic, Actins, Rats, Microscopy, Electron, Thiazoles, rab GTP-Binding Proteins, Latrunculin, Cattle, Carrier Proteins, 030217 neurology & neurosurgery
Abstract

International audience; The GTPase Rab27A interacts with myosin-VIIa and myosin-Va via MyRIP or melanophilin and mediates melanosome binding to actin. Here we show that Rab27A and MyRIP are associated with secretory granules (SGs) in adrenal chromaffin cells and PC12 cells. Overexpression of Rab27A, GTPase-deficient Rab27A-Q78L, or MyRIP reduced secretory responses of PC12 cells. Amperometric recordings of single adrenal chromaffin cells revealed that Rab27A-Q78L and MyRIP reduced the sustained component of release. Moreover, these effects on secretion were partly suppressed by the actin-depolymerizing drug latrunculin but strengthened by jasplakinolide, which stabilizes the actin cortex. Finally, MyRIP and Rab27A-Q78L restricted the motion of SGs in the subplasmalemmal region of PC12 cells, as measured by evanescent-wave fluorescence microscopy. In contrast, the Rab27A-binding domain of MyRIP and a MyRIP construct that interacts with myosin-Va but not with actin increased the mobility of SGs. We propose that Rab27A and MyRIP link SGs to F-actin and control their motion toward release sites through the actin cortex.

Published
2003
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7. Solubilization and Stabilization of Membrane Proteins by Cycloalkane-Modified Amphiphilic Polymers

Author

Baptiste Michon, Christel Le Bon, Fabrice Giusti, Manuela Zoonens, Christophe Tribet, Anaïs Marconnet, Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physico-Chimie des Polymères et des Milieux Dispersés (PPMD), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS) with an '80PRIME' grant, the University Paris-7 (Université de Paris), and the 'Initiative d’Excellence' program from the French State (Grant 'DYNAMO', ANR-11-LABX-0011-01)

Subjects
Polymers and Plastics, Maleic acid, Polymers, [SDV]Life Sciences [q-bio], membrane proteins, Bioengineering, 02 engineering and technology, 010402 general chemistry, amphipols, 01 natural sciences, surfactants, Biomaterials, chemistry.chemical_compound, SMA co-polymers, Escherichia coli, Materials Chemistry, Copolymer, Native state, Acrylic acid, Chemistry, Cycloparaffins, Biological membrane, stability, 021001 nanoscience & nanotechnology, Combinatorial chemistry, 0104 chemical sciences, Membrane, Membrane protein, Styrene maleic anhydride, extraction, 0210 nano-technology, Hydrophobic and Hydrophilic Interactions
Abstract

International audience; Membrane proteins need to be extracted from biological membranes and purified in their native state for most structural and functional in vitro investigations. Amphiphilic copolymers, such as amphipols (APols), have emerged as very useful alternatives to detergents for keeping membrane proteins water-soluble under their native form. However, classical APols, i.e. poly(acrylic acid) (PAA) derivatives, seldom enable direct membrane protein extraction. Poly(styrene-maleic anhydride) copolymers (SMAs), which bear aromatic rings as hydrophobic side groups, have been reported to be more effective extracting agents. In order to test the hypothesis of a role of cyclic hydrophobic moieties in membrane solubilization by copolymers, we have prepared PAA derivatives comprising cyclic rather than linear aliphatic side groups (CyclAPols). As references, APol A8-35, SMAs, and diisobutylene maleic acid (DIBMA) were compared with CyclAPols. Using as models the plasma membrane of Escherichia coli and the extraction-resistant purple membrane from Halobacterium salinarum, we show that CyclAPols combine the extraction efficiency of SMAs with the stabilization afforded to membrane proteins by classical APols such as A8-35.

Published
2020
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8. From the Amelioration of a NADP + ‐dependent Formate Dehydrogenase to the Discovery of a New Enzyme: Round Trip from Theory to Practice

Author

Chiara Lonigro, Marina Simona Robescu, Elisabetta Bergantino, Elisa Beneventi, Laura Cendron, Rudy Rubini, Francesco Filippini, Michele Tavanti, Francesca Zito, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Molecular Biology and Bioinformatics Unit, Department of Biology, Universita degli Studi di Padova, Department of Biology, University of Padova, and Labex Dynamo

Subjects
0106 biological sciences, chemistry.chemical_classification, 0303 health sciences, Chemistry, [SDV]Life Sciences [q-bio], Organic Chemistry, Theory to practice, Formate dehydrogenase, 01 natural sciences, Catalysis, Inorganic Chemistry, 03 medical and health sciences, Enzyme, Biochemistry, 010608 biotechnology, Physical and Theoretical Chemistry, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

International audience

Published
2020
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9. Allosteric modulation of ghrelin receptor signaling by lipids

Author

Louet, Maxime, Casiraghi, Marina, Damian, Marjorie, Costa, Mauricio GS, Renault, Pedro, Gomes, Antoniel AS, Batista, Paulo, M'Kadmi, Céline, Mary, Sophie, Cantel, Sonia, Denoyelle, Severine, Ben Haj Salah, Khoubaib, Perahia, David, Bisch, Paulo, Fehrentz, Jean-Alain, Catoire, Laurent, Floquet, Nicolas, Banères, Jean-Louis, Mustafá, Emilio, Cordisco González, Santiago, Wagner, Renaud, Schiöth, Helgi, Perelló, Mario, Raingo, Jesica, Gomes, Antoniel Augusto Severo, M’Kadmi, Céline, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Universidade Federal do Rio de Janeiro (UFRJ), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), ANR-17-CE11-0011,allosig,allostérie, dynamique conformationnelle et signalisation via les RCPG(2017), ANR-17-CE18-0022,GHScReen2,Biosenseurs originaux pour le criblage des ligands des Récepteurs Couplés aux Protéines G. Application au récepteur de la ghréline.(2017), and Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Phosphatidylinositol 4,5-Diphosphate, MESH: Signal Transduction, MESH: Fluorescence Resonance Energy Transfer, Protein Conformation, Lipid Bilayers, General Physics and Astronomy, MESH: Allosteric Regulation, MESH: Receptors, Ghrelin, MESH: Protein Conformation, 0302 clinical medicine, Fluorescence Resonance Energy Transfer, Receptors, Ghrelin, Receptor, MESH: Lipid Metabolism, 0303 health sciences, Multidisciplinary, Chemistry, MESH: Lipid Bilayers, digestive, oral, and skin physiology, MESH: G(M3) Ganglioside, Lipids, MESH: Phosphatidylinositol 4,5-Diphosphate, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Membrane, Ghrelin, lipids (amino acids, peptides, and proteins), medicine.symptom, Intracellular, Signal Transduction, Biophysical chemistry, MESH: Mutation, G protein, Science, Allosteric regulation, Mechanism of action, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Allosteric Regulation, medicine, G(M3) Ganglioside, Humans, Cysteine, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, Binding Sites, MESH: Humans, Cell Membrane, General Chemistry, MESH: Cysteine, Lipid Metabolism, MESH: Lipids, MESH: Binding Sites, Mutation, Biophysics, 030217 neurology & neurosurgery, MESH: Cell Membrane
Abstract

The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here we demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects. We find that PIP2 shifts the conformational equilibrium of GHSR away from its inactive state, favoring basal and agonist-induced G protein activation. This occurs because of a preferential binding of PIP2 to specific intracellular sites in the receptor active state. Another lipid, GM3, also binds GHSR and favors G protein activation, but mostly in a ghrelin-dependent manner. Finally, we find that not only selective interactions but also the thickness of the bilayer reshapes the conformational repertoire of GHSR, with direct consequences on G protein selectivity. Taken together, this data illuminates the multifaceted role of the membrane components as allosteric modulators of how ghrelin signal could be propagated., The membrane is an integral component of the G protein-coupled receptor signaling machinery. Here authors demonstrate that lipids regulate the signaling efficacy and selectivity of the ghrelin receptor GHSR through specific interactions and bulk effects and observe PIP2 and GM3 induced shifts of the conformational equilibrium of GHSR away from its inactive state.

Published
2021
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10. Cycloalkane-modified amphiphilic polymers provide direct extraction of membrane proteins for CryoEM analysis

Author

Anna J. Higgins, Alex J. Flynn, Anaïs Marconnet, Laura J. Musgrove, Vincent L. G. Postis, Jonathan D. Lippiat, Chun-wa Chung, Tom Ceska, Manuela Zoonens, Frank Sobott, Stephen P. Muench, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Polymers, QH301-705.5, [SDV]Life Sciences [q-bio], Medicine (miscellaneous), macromolecular substances, 02 engineering and technology, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, detergent, Cryoelectron microscopy, SMA co-polymers, Escherichia coli, membrane protein, Biology (General), Author Correction, 030304 developmental biology, 0303 health sciences, Membranes, Escherichia coli Proteins, amphipol, Cycloparaffins, 021001 nanoscience & nanotechnology, Multidrug Resistance-Associated Proteins, 0210 nano-technology, General Agricultural and Biological Sciences, CryoEM
Abstract

Membrane proteins are essential for cellular growth, signalling and homeostasis, making up a large proportion of therapeutic targets. However, the necessity for a solubilising agent to extract them from the membrane creates challenges in their structural and functional study. Although amphipols have been very effective for single-particle electron cryo-microscopy (cryoEM) and mass spectrometry, they rely on initial detergent extraction before exchange into the amphipol environment. Therefore, circumventing this pre-requirement would be a big advantage. Here we use an alternative type of amphipol: a cycloalkane-modified amphiphile polymer (CyclAPol) to extract Escherichia coli AcrB directly from the membrane and demonstrate that the protein can be isolated in a one-step purification with the resultant cryoEM structure achieving 3.2 Å resolution. Together this work shows that cycloalkane amphipols provide a powerful approach for the study of membrane proteins, allowing native extraction and high-resolution structure determination by cryoEM., Higgins et al. present a cycloalkane-modified amphiphilic polymer that can provide direct extraction of membrane proteins for Cryo-EM analysis. They show its utility by extracting and solving the structure of AcrB to a high resolution of 3.2 Å by single particle cryo-EM.

Published
2021
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11. A large disordered region confers a wide spanning volume to vertebrate Suppressor of Fused as shown in a trans-species solution study

Author

Jabrani A, Matthieu Sanial, Biou, Marc Baaden, Makamte S, Thureau A, Paquelin A, Anne Plessis, Roudenko O, Francesco Oteri, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
0303 health sciences, Circular dichroism, biology, Chemistry, [SDV]Life Sciences [q-bio], 030302 biochemistry & molecular biology, Vertebrate, biology.organism_classification, Phenotype, Cell biology, Repressor Proteins, 03 medical and health sciences, Structural Biology, biology.animal, Animals, Drosophila, Hedgehog Proteins, Psychological repression, Zebrafish, Hedgehog, Function (biology), 030304 developmental biology, Sequence (medicine), Signal Transduction
Abstract

Hedgehog (Hh) pathway inhibition by the conserved protein Suppressor of Fused (SuFu) is crucial to vertebrate development. By constrast, SuFu loss-of-function mutant has little effect in drosophila. Previous publications showed that the crystal structures of human and drosophila SuFu consist of two ordered domains that are capable of breathing motions upon ligand binding. However, the crystal structure of human SuFu does not give information about twenty N-terminal residues (IDR1) and an eighty-residue-long region predicted as disordered (IDR2) in the C-terminus, whose function is important for the pathway repression. These two intrinsically disordered regions (IDRs) are species-dependent. To obtain information about the IDR regions, we studied full-length SuFu's structure in solution, both with circular dichroism and small angle X-ray scattering, comparing drosophila, zebrafish and human species, to better understand this considerable difference. Our studies show that, in spite of similar crystal structures restricted to ordered domains, drosophila and vertebrate SuFu have very different structures in solution. The IDR2 of vertebrates spans a large area, thus enabling it to reach for partners and be accessible for post-translational modifications. Furthermore, we show that the IDR2 region is highly conserved within phyla but varies in length and sequence, with insects having a shorter disordered region while that of vertebrates is broad and mobile. This major variation may explain the different phenotypes observed upon SuFu removal.

Published
2021

12. Functional and structural characterization of Serratia marcescens ExbB: determinants of the interaction with HasB/TonB

Author

Biou, Valérie, Diogo Adaixo, Ricardo Jorge, Chami, Mohamed, Coureux, Pierre-Damien, Laurent, Benoist, Ntsogo, Yvette, de Amorim, Gisele Cardoso, Izadi-Pruneyre, Nadia, Malosse, Christian, Chamot-Rooke, Julia, Stahlberg, Henning, Delepelaire, Philippe, CNRS UMR 7099, UMR 7099, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
[SDV]Life Sciences [q-bio], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology
Abstract

Summary ExbBD is part of a cytoplasmic membrane molecular motor driven by the proton-motive force. It belongs to the larger family of motors involved in nutriment import across the outer membrane of Gram-negative bacteria (ExbBD), flagellar rotation (MotAB) or late steps of cell division in Gram-negative bacteria (TolQR). ExbB and ExbD are integral membrane proteins with three (ExbB) or one (ExbD) transmembrane segment. Here we have solved by single-particle cryo-EM the structures of ExbB alone and of the ExbB-ExbD complex of the opportunistic pathogen Serratia marcescens . ExbB Sm alone behaves as a stable pentamer, and the complex displays the ExbB 5 -ExbD 2 stoichiometry. This is similar to what has been observed for ExbB-ExbD complexes from Escherichia coli and Pseudomonas savastanoi as well as MotAB complexes from various species. We identified residues located in the first TM of ExbB Sm and ExbB Ec that are likely involved in the interaction with TonB/HasB and that are essential for function. ExbB Sm has a ca. 40 residues long periplasmic extension absent in E. coli . Such long ExbB’s are found in some Gammaproteobacteria, and several genera of Alphaproteobacteria. We show that this extension interacts with HasB, a dedicated TonB paralog from the heme acquisition system (Has) from S. marcescens . We also show that it is involved in heme acquisition via the Has system from S. marcescens . ExbB Sm represents thus a new class of ExbB protein and our results shed light on the specificity determinants between the ExbB-ExbD complex and their associated TonB partners.

Published
2021

13. Concerted conformational dynamics and water movements in the ghrelin G protein-coupled receptor

Author

Laurent J. Catoire, Sonia Cantel, Marjorie Damian, Jean-Alain Fehrentz, Maxime Louet, Antoniel As Gomes, Jean-Louis Banères, Sophie Mary, Paulo R. Batista, David Perahia, Paulo Mascarello Bisch, Mauricio Gs Costa, Khoubaib Ben Haj Salah, Céline M'Kadmi, Marina Casiraghi, Pedro Renault, Severine Denoyelle, Nicolas Floquet, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Department of Molecular and Cellular Physiology [Stanford], Stanford Medicine, Stanford University-Stanford University, Laboratoire de biologie et pharmacologie appliquée (LBPA), Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Ecole Normale Supérieure Paris-Saclay (ENS Paris Saclay), Universidade Federal do Rio de Janeiro (UFRJ), This work was supported by CNRS, Université de Montpellier, Agence Nationale de la Recherche (ANR-17-CE11-0011, ANR-17-CE11-22, ANR-17-CE18-0022), EpiGenMed Labex (post-doctoral fellowship to KBH) and DYNAMO Labex (post-doctoral fellowship to MC). This programreceived funding from the European Union’s Horizon 2020 research and innovation programmeunder the Marie Sklodowska-Curie grant agreement n˚ 799376. Mass spectrometry analyses wereperformed on the instruments located in the IBMM platform of instrumentation, Laboratoire deMesures Physiques (LMP) of Université de Montpellier. We thank GENCI (Grand EquipementNational de Calcul Intensif), CINES (Centre Informatique National de l’Enseignement Supérieur), and IDRIS (Institut du développement et des ressources en informatique scientifique) for computational, ANR-17-CE11-0011,allosig,allostérie, dynamique conformationnelle et signalisation via les RCPG(2017), ANR-17-CE11-0022,GPCteR,Mécanismes moléculaires des régions C-terminales désordonnées et fonctionnelles des RCPG et impact sur les voies de la signalisation cellulaire dépendantes de l'arrestine(2017), and Centre National de la Recherche Scientifique (CNRS)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)

Subjects
QH301-705.5, Science, Growth hormone secretagogue receptor, Chemical biology, chemical biology, 010402 general chemistry, Ligands, 01 natural sciences, General Biochemistry, Genetics and Molecular Biology, Receptors, G-Protein-Coupled, 03 medical and health sciences, Molecular dynamics, GPCR, Biochemistry and Chemical Biology, biochemistry, Humans, Biology (General), Receptor, Receptors, Ghrelin, 030304 developmental biology, G protein-coupled receptor, chemistry.chemical_classification, 0303 health sciences, General Immunology and Microbiology, [SDV.BA]Life Sciences [q-bio]/Animal biology, General Neuroscience, digestive, oral, and skin physiology, E. coli, General Medicine, Ghrelin, 0104 chemical sciences, Amino acid, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Transmembrane domain, chemistry, Biophysics, Medicine, signaling, hydration, Research Article, Human, Signal Transduction
Abstract

International audience; There is increasing support for water molecules playing a role in signal propagation through G protein-coupled receptors (GPCRs). However, exploration of the hydration features of GPCRs is still in its infancy. Here, we combined site-specific labeling with unnatural amino acids to molecular dynamics to delineate how local hydration of the ghrelin receptor growth hormone secretagogue receptor (GHSR) is rearranged upon activation. We found that GHSR is characterized by a specific hydration pattern that is selectively remodeled by pharmacologically distinct ligands and by the lipid environment. This process is directly related to the concerted movements of the transmembrane domains of the receptor. These results demonstrate that the conformational dynamics of GHSR are tightly coupled to the movements of internal water molecules, further enhancing our understanding of the molecular bases of GPCR-mediated signaling.

Published
2021
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14. How to best estimate the viscosity of lipid bilayers

Author

Gamal Rayan, Martin Picard, Wladimir Urbach, Vladimir Adrien, Isabelle Broutin, Ksenia Astafyeva, Nicolas Taulier, Patrick F.J. Fuchs, Laboratoire de physique de l'ENS - ENS Paris (LPENS), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Département de Physique de l'ENS-PSL, École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL), Cibles Thérapeutiques et conception de médicaments (CiTCoM - UMR 8038), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire des biomolécules (LBM UMR 7203), Chimie Moléculaire de Paris Centre (FR 2769), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-École normale supérieure - Paris (ENS-PSL), Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Département de Chimie - ENS Paris, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Mécanismes Moléculaires Membranaires, Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)-Département de Physique de l'ENS-PSL, Laboratoire d'Imagerie Biomédicale (LIB), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Taulier, Nicolas, Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)-Sorbonne Université (SU)-École normale supérieure - Paris (ENS Paris), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Chimie Moléculaire de Paris Centre (FR 2769), Institut de Chimie du CNRS (INC)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de physique de l'ENS - ENS Paris (LPENS (UMR_8023)), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
Fluorescence-lifetime imaging microscopy, FLIM, Diffusion, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Lipid Bilayers, Biophysics, 02 engineering and technology, 010402 general chemistry, 01 natural sciences, Biochemistry, lipids, Viscosity, Lipid bilayer, Molecular diffusion, [PHYS.PHYS.PHYS-BIO-PH] Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], Chemistry, Bilayer, Organic Chemistry, Fluorescence recovery after photobleaching, 021001 nanoscience & nanotechnology, 0104 chemical sciences, Membrane, Microscopy, Fluorescence, Chemical physics, FRAP, Hydrodynamics, fluorescence, 0210 nano-technology, bilayers
Abstract

The viscosity of lipid bilayers is a property relevant to biological function, as it affects the diffusion of membrane macromolecules. To determine its value, and hence portray the membrane, various literature-reported techniques lead to significantly different results. Herein we compare the results issuing from two widely used techniques to determine the viscosity of membranes: the Fluorescence Lifetime Imaging Microscopy (FLIM), and Fluorescence Recovery After Photobleaching (FRAP). FLIM relates the time of rotation of a molecular rotor inserted into the membrane to the macroscopic viscosity of a fluid. Whereas FRAP measures molecular diffusion coefficients. This approach is based on a hydrodynamic model connecting the mobility of a membrane inclusion to the viscosity of the membrane. We show that: - The first method is very sensitive to local changes in viscosity; however, most often it would only provide the viscosity of the hydrophobic part of the membrane. - The membrane viscosity is adequately estimated when the hydrodynamic model approach is applied to the mobility of micrometric size membrane inclusion but not for nanometric size inclusions such as lipids or proteins. In this case, the calculated value extracted from the same hydrodynamic model characterizes the interaction of the given nano-inclusion with the bilayer instead of the bilayer viscosity. This article emphasizes the pitfalls to be avoided and the rules to be observed in order to obtain a value of the bilayer viscosity that characterizes the bilayer instead of interactions between the bilayer and the embedded probe.

Published
2021
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15. Full‐length G glycoprotein directly extracted from rabies virus with detergent and then stabilized by amphipols in liquid and freeze‐dried forms

Author

Benoit Strobbe, Christel Le Bon, Aure Saulnier, Léna Clavier, Didier Clénet, Manuela Zoonens, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Antigenicity, [SDV]Life Sciences [q-bio], Detergents, G glycoprotein from rabies virus, Bioengineering, medicine.disease_cause, 030226 pharmacology & pharmacy, Applied Microbiology and Biotechnology, Virus, stable freeze-dried A8-35 formulation, A8-35 formulation study, 03 medical and health sciences, 0302 clinical medicine, Antigen, Viral Envelope Proteins, Amphiphile, medicine, Pathogen, Antigens, Viral, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Protein Stability, Rabies virus, Freeze Drying, chemistry, Membrane protein, Biochemistry, amphipol-stabilized integral membrane protein, Glycoprotein, Biotechnology
Abstract

Pathogen surface antigens are at the forefront of the viral strategy when invading host organisms. These antigens, including membrane proteins (MPs), are broadly targeted by the host immune response. Obtaining these MPs in a soluble and stable form constitutes a real challenge, regardless of the application purposes (e.g. quantification/characterization assays, diagnosis, preventive and curative strategies). A rapid process to obtain a native-like antigen by solubilization of a full-length MP directly from a pathogen is reported herein. Rabies virus (RABV) was used as a model for this demonstration and its full-length G glycoprotein (RABV-G) was stabilized with amphipathic polymers, named amphipols (APols). The stability of RABV-G trapped in APol A8-35 (RABV-G/A8-35) was evaluated under different stress conditions (temperature, agitation and light exposure). RABV-G/A8-35 in liquid form exhibited higher unfolding temperature (+ 6°C) than in detergent and was demonstrated to be antigenically stable over one month at 5°C and 25°C. Kinetic modeling of antigenicity data predicted an antigenic stability of RABV-G/A8-35 in solution of up to one year at 5°C. The RABV-G/A8-35 complex formulated in an optimized buffer composition and subsequently freeze-dried displayed a long-term stability for 2-years at 5°C, 25°C and 37°C. This study reports for the first time that a natural full-length MP extracted from a virus, complexed to APols and subsequently freeze-dried, displayed long-term antigenic stability, without requiring storage under refrigerated conditions. This article is protected by copyright. All rights reserved.

Published
2021
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16. Binding of HasA by its transmembrane receptor HasR follows a conformational funnel mechanism

Author

Audrey Boniface-Guiraud, Stefanie Becker, Philippe Delepelaire, Simon Becker, Kay Diederichs, Thomas E. Exner, Wolfram Welte, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Fachbereich Biologie, and University of Konstanz

Subjects
0301 basic medicine, Heme acquisition system, business.product_category, Mutant, Biophysics, Receptors, Cell Surface, Heme, Molecular Dynamics Simulation, 01 natural sciences, 03 medical and health sciences, chemistry.chemical_compound, Molecular dynamics, Bacterial Proteins, ddc:570, HasR, 0103 physical sciences, Receptor, ComputingMilieux_MISCELLANEOUS, Serratia marcescens, Physics, Binding Sites, 010304 chemical physics, HasA, Crystal structure, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, Periplasmic space, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Transmembrane protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Outer membrane, 030104 developmental biology, chemistry, Mutation, Original Article, Funnel, Bacterial outer membrane, business, Carrier Proteins, Protein Binding
Abstract

HasR in the outer membrane of Serratia marcescens binds secreted, heme-loaded HasA and translocates the heme to the periplasm to satisfy the cell’s demand for iron. The previously published crystal structure of the wild-type complex showed HasA in a very specific binding arrangement with HasR, apt to relax the grasp on the heme and assure its directed transfer to the HasR-binding site. Here, we present a new crystal structure of the heme-loaded HasA arranged with a mutant of HasR, called double mutant (DM) in the following that seemed to mimic a precursor stage of the abovementioned final arrangement before heme transfer. To test this, we performed first molecular dynamics (MD) simulations starting at the crystal structure of the complex of HasA with the DM mutant and then targeted MD simulations of the entire binding process beginning with heme-loaded HasA in solution. When the simulation starts with the former complex, the two proteins in most simulations do not dissociate. When the mutations are reverted to the wild-type sequence, dissociation and development toward the wild-type complex occur in most simulations. This indicates that the mutations create or enhance a local energy minimum. In the targeted MD simulations, the first protein contacts depend upon the chosen starting position of HasA in solution. Subsequently, heme-loaded HasA slides on the external surface of HasR on paths that converge toward the specific arrangement apt for heme transfer. The targeted simulations end when HasR starts to relax the grasp on the heme, the subsequent events being in a time regime inaccessible to the available computing power. Interestingly, none of the ten independent simulation paths visits exactly the arrangement of HasA with HasR seen in the crystal structure of the mutant. Two factors which do not exclude each other could explain these observations: the double mutation creates a non-physiologic potential energy minimum between the two proteins and /or the target potential in the simulation pushes the system along paths deviating from the low-energy paths of the native binding processes. Our results support the former view, but do not exclude the latter possibility. Electronic supplementary material The online version of this article (10.1007/s00249-019-01411-1) contains supplementary material, which is available to authorized users.

Published
2019
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17. Structure, Assembly, and Function of Tripartite Efflux and Type 1 Secretion Systems in Gram-Negative Bacteria

Author

Ilyas Alav, Martin Picard, Vassiliy N. Bavro, Miriam S. Kuth, Jessica Kobylka, Jessica M A Blair, Klaas M. Pos, Membrane Transport Machinerie, Goethe-University Frankfurt am Main, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
Gram-negative bacteria, Protein Conformation, [SDV]Life Sciences [q-bio], Computational biology, Review, Molecular Dynamics Simulation, 010402 general chemistry, 01 natural sciences, Structure-Activity Relationship, Gram-Negative Bacteria, Inner membrane, Type I Secretion Systems, biology, 010405 organic chemistry, Chemistry, Signal transducing adaptor protein, Lipid bilayer fusion, Membrane Transport Proteins, General Chemistry, Periplasmic space, biology.organism_classification, 0104 chemical sciences, Structural biology, ATP-Binding Cassette Transporters, Efflux, Bacterial outer membrane, Bacterial Outer Membrane Proteins
Abstract

International audience; Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal componentsthe energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS-and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.

Published
2021
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18. Folding Control in the Path of Type 5 Secretion

Author

Nathalie Dautin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
folding, Protein Folding, two-partner secretion, Type V Secretion Systems, Virulence Factors, [SDV]Life Sciences [q-bio], Health, Toxicology and Mutagenesis, intimin, Virulence, Review, Biology, Toxicology, type 5 secretion system, Bacterial cell structure, 03 medical and health sciences, trimeric autotransporter, Gram-Negative Bacteria, Secretion, 030304 developmental biology, 0303 health sciences, 030306 microbiology, Mechanism (biology), autotransporter, Cell biology, Folding (chemistry), Bacterial adhesin, secretion, Medicine, Protein folding, invasin, Biogenesis
Abstract

International audience; The type 5 secretion system (T5SS) is one of the more widespread secretion systems in Gram-negative bacteria. Proteins secreted by the T5SS are functionally diverse (toxins, adhesins, enzymes) and include numerous virulence factors. Mechanistically, the T5SS has long been considered the simplest of secretion systems, due to the paucity of proteins required for its functioning. Still, despite more than two decades of study, the exact process by which T5SS substrates attain their final destination and correct conformation is not totally deciphered. Moreover, the recent addition of new sub-families to the T5SS raises additional questions about this secretion mechanism. Central to the understanding of type 5 secretion is the question of protein folding, which needs to be carefully controlled in each of the bacterial cell compartments these proteins cross. Here, the biogenesis of proteins secreted by the Type 5 secretion system is discussed, with a focus on the various factors preventing or promoting protein folding during biogenesis.

Published
2021
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19. Quantitative real-time analysis of the efflux by the MacAB-TolC tripartite efflux pump clarifies the role of ATP hydrolysis within mechanotransmission mechanism

Author

William Batista dos Santos, Laurent J. Catoire, Martin Picard, Vassiliy N. Bavro, Dhenesh Puvanendran, Quentin Cece, Hager Souabni, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), University of Essex, and ANR-16-CE11-0001,inVIVE,Bases moléculaires du transport actif d'antibiotiques par la pompe d'efflux MexA-MexB-OprM de Pseudomonas aeruginosa. in vitro veritas(2016)

Subjects
QH301-705.5, Biophysics, Medicine (miscellaneous), Context (language use), Biochemistry, Article, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Adenosine Triphosphate, 0302 clinical medicine, ATP hydrolysis, Escherichia coli, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Nucleotide, Biology (General), 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Escherichia coli Proteins, Hydrolysis, Membrane Transport Proteins, Substrate (chemistry), Transporter, chemistry, Membrane protein, ATP-Binding Cassette Transporters, Efflux, General Agricultural and Biological Sciences, Energy source, 030217 neurology & neurosurgery, Bacterial Outer Membrane Proteins
Abstract

Tripartite efflux pumps built around ATP-binding cassette (ABC) transporters are membrane protein machineries that perform vectorial export of a large variety of drugs and virulence factors from Gram negative bacteria, using ATP-hydrolysis as energy source. Determining the number of ATP molecules consumed per transport cycle is essential to understanding the efficiency of substrate transport. Using a reconstituted pump in a membrane mimic environment, we show that MacAB-TolC from Escherichia coli couples substrate transport to ATP-hydrolysis with high efficiency. Contrary to the predictions of the currently prevailing “molecular bellows” model of MacB-operation, which assigns the power stroke to the ATP-binding by the nucleotide binding domains of the transporter, by utilizing a novel assay, we report clear synchronization of the substrate transfer with ATP-hydrolysis, suggesting that at least some of the power stroke for the substrate efflux is provided by ATP-hydrolysis. Our findings narrow down the window for energy consumption step that results in substrate transition into the TolC-channel, expanding the current understanding of the efflux cycle of the MacB-based tripartite assemblies. Based on that we propose a modified model of the MacB cycle within the context of tripartite complex assembly., Souabni et al. introduce a new analytical method for the study of ABC tripartite efflux pumps, by using a lipid scaffold that mimics the protein’s native environment and a spectroscopic assay, employing quantum dots (QD), that monitors ATP hydrolysis and substrate transport in real-time. When applied to MacAB-TolC tripartite pump complex, results suggest high coupling efficiency between transport and energy consumption in the system.

Published
2021
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20. Amphipathic environments for determining the structure of membrane proteins by single-particle electron cryo-microscopy

Author

Christel Le Bon, Manuela Zoonens, Baptiste Michon, Jean-Luc Popot, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre National de la Recherche Scientifique (CNRS), and The Centre National de la Recherche Scientifique (CNRS), including specific '80PRIME' grant, the Université de Paris (Université Paris-7), and the 'Initiative d’Excellence' program from the French State (Grant 'DYNAMO', ANR-11-LABX-0011-01)

Subjects
0301 basic medicine, Materials science, integral membrane protein complexes, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Cryoelectron Microscopy, Biophysics, Membrane Proteins, Nanotechnology, Electrons, amphipols, lipids, 03 medical and health sciences, Surface-Active Agents, 030104 developmental biology, 0302 clinical medicine, Membrane protein, Structural biology, detergents, nanodiscs, Microscopy, Amphiphile, structural biology, Particle, Sample preparation, 030217 neurology & neurosurgery
Abstract

Over the past decade, the structural biology of membrane proteins (MPs) has taken a new turn thanks to epoch-making technical progress in single-particle electron cryo-microscopy (cryo-EM) as well as to improvements in sample preparation. The present analysis provides an overview of the extent and modes of usage of the various types of surfactants for cryo-EM studies. Digitonin, dodecylmaltoside, protein-based nanodiscs, lauryl maltoside-neopentyl glycol, glyco-diosgenin, and amphipols (APols) are the most popular surfactants at the vitrification step. Surfactant exchange is frequently used between MP purification and grid preparation, requiring extensive optimization each time the study of a new MP is undertaken. The variety of both the surfactants and experimental approaches used over the past few years bears witness to the need to continue developing innovative surfactants and optimizing conditions for sample preparation. The possibilities offered by novel APols for EM applications are discussed.

Published
2021
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21. Study of membrane deformations induced by Hepatitis C protein NS4B and its terminal amphipathic peptides

Author

Ouldali, Malika, Moncoq, Karine, de la Croix de la Valette, Agnès, Arteni, Ana, Betton, Jean-Michel, Lepault, Jean, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Virologie Structurale, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Biochimie Structurale, Agence National contre le Sida et l'Hépatite C (ANRS), Agence National pour la Recherche (ANR), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris]

Subjects
HCV-NS4B induced membrane disorder, NS4B peptides induced membrane perforation, viruses, [SDV]Life Sciences [q-bio], Cryo-EM of membranes disordered by NS4B, CryoEM of membranes fused by NS4B peptide, One NS4B peptide induced membrane fusion
Abstract

International audience; Many viruses destabilize cellular membranous compartments to form their replication complexes, but the mechanism(s) underlying membrane perturbation remains unknown. Expression in eukaryotic cells of NS4B, a protein of the hepatitis C virus (HCV), alters membranous complexes and induces structures similar to the so-called membranous web that appears crucial to the formation of the HCV replication complex. As over-expression of the protein is lethal to both prokaryotic and eukaryotic cells, NS4B was produced in large quantities in a "cell-free" system in the presence of detergent, after which it was inserted into lipid membranes. X-ray diffraction revealed that NS4B modifies the phase diagram of synthetic lipid aqueous phases considerably, perturbing the transition temperature and cooperativity. Cryo-electron microscopy demonstrated that NS4B introduces significant disorder in the synthetic membrane as well as discontinuities that could be interpreted as due to the formation of pores and membrane merging events. C- and N-terminal fragments of NS4B are both able to destabilize liposomes. While most NS4B amphipathic peptides perforate membranes, one NS4B peptide induces membrane fusion. Cryo-electron microscopy reveals a particular structure that can be interpreted as arising from hemi-fusion-like events. Amphipathic domains are present in many proteins, and if exposed to the aqueous cytoplasmic medium are sufficient to destabilize membranes in order to form viral replication complexes. These domains have important functions in the viral replication cycle, and thus represent potential targets for the development of anti-viral molecules.

Published
2021
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22. Structure of the agonist 12–HHT in its BLT2 receptor-bound state

Author

Christel Le Bon, Jutta Rieger, Karine Moncoq, Marjorie Damian, Elodie Point, Jean-Louis Banères, Alexandre Pozza, Marina Casiraghi, Fabrice Giusti, Laurent J. Catoire, Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Chimie des polymères (LCP), Institut Parisien de Chimie Moléculaire (IPCM), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Unité de Chimie et Procédés (UCP), École Nationale Supérieure de Techniques Avancées (ENSTA Paris), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut Parisien de Chimie Moléculaire (IPCM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC (FR_550)), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Institut de Chimie du CNRS (INC)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Agonist, Leukotriene B4, medicine.drug_class, Molecular Conformation, Receptors, Leukotriene B4, lcsh:Medicine, Ligand, Cell surface receptor, Ligands, Article, 03 medical and health sciences, chemistry.chemical_compound, Docking (dog), Stereochemistry, medicine, Humans, Homology modeling, lcsh:Science, Receptor, Nuclear Magnetic Resonance, Biomolecular, Unsaturated fatty acid, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Multidisciplinary, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Leukotriene receptor, lcsh:R, 030302 biochemistry & molecular biology, Molecular Docking Simulation, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [CHIM.POLY]Chemical Sciences/Polymers, Docking (molecular), Biophysics, Fatty Acids, Unsaturated, lcsh:Q, Solution-state NMR, [CHIM.CHEM]Chemical Sciences/Cheminformatics, Protein Binding
Abstract

G Protein-Coupled receptors represent the main communicating pathway for signals from the outside to the inside of most of eukaryotic cells. They define the largest family of integral membrane receptors at the surface of the cells and constitute the main target of the current drugs on the market. The low affinity leukotriene receptor BLT2 is a receptor involved in pro- and anti-inflammatory pathways and can be activated by various unsaturated fatty acid compounds. We present here the NMR structure of the agonist 12–HHT in its BLT2-bound state and a model of interaction of the ligand with the receptor based on a conformational homology modeling associated with docking simulations. Put into perspective with the data obtained with leukotriene B4, our results illuminate the ligand selectivity of BLT2 and may help define new molecules to modulate the activity of this receptor.

Published
2020
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23. Etudes par SAXS et simulations de dynamiques moléculaires des propriétés structurales sur le récepteur membranaire ShuA dans les micelles DDM et dans un modèle de membrane externe de bactéries Gram-négatives

Author

Abel, Stéphane, Marchi, Massimo, Solier, Justine, Finet, Stéphanie, Brillet, Karl, Bonnete, Françoise, Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Service de Bioénergétique, Biologie Stucturale, et Mécanismes (SB2SM), Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de minéralogie et de physique des milieux condensés (IMPMC), Université Pierre et Marie Curie - Paris 6 (UPMC)-IPG PARIS-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Architecture et Réactivité de l'ARN (ARN), Institut de biologie moléculaire et cellulaire (IBMC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), ANR-14-LAB7-0002,CHEM2STAB,Nouvelles Approches Moléculaires pour l'isolement de Protéines Membranaires(2014), ANR-10-LABX-0005,CheMISyst,CHEmistry of Molecular and Interfacial Systems(2010), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Université Paris Diderot - Paris 7 (UPD7)-Institut de Physique du Globe de Paris (IPG Paris)-Centre National de la Recherche Scientifique (CNRS), and Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
DDM, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, SEC-MALLS, Molecular dynamics simulations, Membrane transporter ShuA, SEC-SAXS, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Outer membrane model
Abstract

International audience

Published
2020
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24. Inducible Intracellular Membranes: Molecular Aspects and Emerging Applications

Author

Christophe Tribet, Jorge Royes, Valérie Biou, Bruno Miroux, Nathalie Dautin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0106 biological sciences, Lipid biosynthesis, Protein Conformation, [SDV]Life Sciences [q-bio], lcsh:QR1-502, Bioengineering, Review, Membrane curvature, 01 natural sciences, Applied Microbiology and Biotechnology, lcsh:Microbiology, 03 medical and health sciences, chemistry.chemical_compound, 010608 biotechnology, Cardiolipin, Inner membrane, Phospholipids, 030304 developmental biology, Organelles, 0303 health sciences, Membrane biosynthesis, Cell Membrane, microbiology, Membrane Proteins, Membrane remodeling, Cell biology, Membrane, chemistry, Membrane protein, Membrane biogenesis, Cell Surface Extensions, Intracellular, Biotechnology
Abstract

Membrane remodeling and phospholipid biosynthesis are normally tightly regulated to maintain the shape and function of cells. Indeed, different physiological mechanisms ensure a precise coordination between de novo phospholipid biosynthesis and modulation of membrane morphology. Interestingly, the overproduction of certain membrane proteins hijack these regulation networks, leading to the formation of impressive intracellular membrane structures in both prokaryotic and eukaryotic cells. The proteins triggering an abnormal accumulation of membrane structures inside the cells (or membrane proliferation) share two major common features: (1) they promote the formation of highly curved membrane domains and (2) they lead to an enrichment in anionic, cone-shaped phospholipids (cardiolipin or phosphatidic acid) in the newly formed membranes. Taking into account the available examples of membrane proliferation upon protein overproduction, together with the latest biochemical, biophysical and structural data, we explore the relationship between protein synthesis and membrane biogenesis. We propose a mechanism for the formation of these non-physiological intracellular membranes that shares similarities with natural inner membrane structures found in α-proteobacteria, mitochondria and some viruses-infected cells, pointing towards a conserved feature through evolution. We hope that the information discussed in this review will give a better grasp of the biophysical mechanisms behind physiological and induced intracellular membrane proliferation, and inspire new applications, either for academia (high-yield membrane protein production and nanovesicle production) or industry (biofuel production and vaccine preparation).

Published
2020
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25. Biotinylated non-ionic amphipols for GPCR ligands screening

Author

Françoise Bonneté, Marjorie Damian, Jean-Alain Fehrentz, Jean-Louis Banères, Pierre Guillet, Michaël Bosco, Mélanie Roche, Ange Polidori, Grégory Durand, Vinay Chauhan, Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Avignon Université (AU), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), ANR-17-CE18- 0022, ANR-10-BLAN-1535, ANR-17-CE18-0022,GHScReen2,Biosenseurs originaux pour le criblage des ligands des Récepteurs Couplés aux Protéines G. Application au récepteur de la ghréline.(2017), and ANR-10-BLAN-1535,X-Or,Orthèses moléculaires favorisant la cristallisation de protéines membranaires. Application à la détermination structurale de pompes d'efflux multi-drogues(2010)

Subjects
Magnetic Resonance Spectroscopy, Polymers, [SDV]Life Sciences [q-bio], Biotin, 7. Clean energy, General Biochemistry, Genetics and Molecular Biology, Polymerization, Receptors, G-Protein-Coupled, Methylamines, 03 medical and health sciences, chemistry.chemical_compound, X-Ray Diffraction, Dynamic light scattering, Scattering, Small Angle, Amphiphile, Humans, Biotinylation, Colloids, Sulfhydryl Compounds, Receptors, Ghrelin, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, biology, Ligand, 030302 biochemistry & molecular biology, Polymer, Combinatorial chemistry, Dynamic Light Scattering, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, HEK293 Cells, Monomer, [CHIM.POLY]Chemical Sciences/Polymers, Acrylates, chemistry, biology.protein, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Streptavidin, Avidin
Abstract

International audience; We present herein the synthesis of biotin-functionalized polymers (BNAPols) that have been developed for the fixation of membrane proteins (MPs) onto surfaces. BNAPols were synthesized by free-radical polymerization of a tris(hydroxymethyl)acrylamidomethane (THAM)-derived amphiphilic monomer in the presence of a thiol-based transfer agent with an azido group. Then a Huisgen-cycloaddition reaction was performed with Biotin-(PEG) 8-alkyne that resulted in formation of the biotinylated polymers. The designed structure of BNAPols was confirmed by NMR spectroscopy, and a HABA/avidin assay was used for estimating the percentage of biotin grafted on the polymer end chain. The colloidal characterization of these biotin-functionalized polymers was done using both dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) techniques. These BNAPols were used to stabilize a model G protein-coupled receptor (GPCR), the human Growth Hormone Secretagogue Receptor (GHSR), out of its membrane environment. Subsequent immobilization of the BNAPols:GHSR complex onto a streptavidin-coated surface allowed screening of ligands based both on their ability to bind the immobilized receptor and to trigger GHSR conformational changes using the fluorescence energy transfer (FRET)-based assay. This opens the way to the use of biotinylated NAPols to immobilize functional, unmodified, membrane proteins, providing original sensor devices for multiple applications including innovative ligand screening assays.

Published
2020
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26. Quantum dot probes for the quantitative study of drug transport by the MacAB TolC efflux pump in lipid scaffolds

Author

D. Puvanendran, Martin Picard, Hager Souabni, Quentin Cece, W. Batista dos Santos, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Skirball Institute of Biomolecular Medicine, New York University [New York] (NYU), and NYU System (NYU)-NYU System (NYU)

Subjects
0303 health sciences, Gram-negative bacteria, biology, Chemistry, [SDV]Life Sciences [q-bio], Virulence, Substrate (chemistry), Transporter, Membrane protein reconstitution, biology.organism_classification, 03 medical and health sciences, 0302 clinical medicine, ABC ATP-binding cassette, Membrane protein, ATP hydrolysis, Quantum dot, Biophysics, Efflux, 030217 neurology & neurosurgery, 030304 developmental biology
Abstract

ABC tripartite efflux pumps are macromolecular membrane protein machineries that expel a large variety of drugs and export virulence factors from Gram negative bacteria. Using a lipid scaffold mimicking the two-membrane environment of the transporter and designing spectroscopic conditions allowing the monitoring of both ATP hydrolysis and substrate transport in real time, we show that MacAB-TolC accommodates transport and energy consumption with high coupling efficiency.

Published
2020

27. Inhibition of patched drug efflux increases vemurafenib effectiveness against resistant braf(V600E) melanoma

Author

Signetti, Laurie, Elizarov, Nelli, Simsir, Méliné, Paquet, Agnès, Douguet, Dominique, Labbal, Fabien, Debayle, Delphine, Di Giorgio, Audrey, Biou, Valérie, Girard, Christophe, Duca, Maria, Bretillon, Lionel, Bertolotto, Corine, Verrier, Bernard, Azoulay, Stéphane, Mus-Veteau, Isabelle, Institut de pharmacologie moléculaire et cellulaire (IPMC), Centre National de la Recherche Scientifique (CNRS)-Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA), Institut de Chimie de Nice (ICN), Université Nice Sophia Antipolis (... - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Centre méditerranéen de médecine moléculaire (C3M), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Côte d'Azur (UCA), Centre des Sciences du Goût et de l'Alimentation [Dijon] (CSGA), Université de Bourgogne (UB)-AgroSup Dijon - Institut National Supérieur des Sciences Agronomiques, de l'Alimentation et de l'Environnement-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Université Bourgogne Franche-Comté [COMUE] (UBFC), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie des Molécules Bioactives et des Arômes (LCMBA), CNRS prematuration programme, SATT-Sud-Est and the association France Cancer., ANR-15-IDEX-0001,UCA JEDI,Idex UCA JEDI(2015), ANR-10-LABX-0011,Entreprendre,Entrepreneurship(2010), Julien, Sabine, Idex UCA JEDI - - UCA JEDI2015 - ANR-15-IDEX-0001 - IDEX - VALID, Laboratoires d'excellence - Entrepreneurship - - Entreprendre2010 - ANR-10-LABX-0011 - LABX - VALID, Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS)-Université Côte d'Azur (UCA), and Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité)

Subjects
[SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, Patched, chemotherapy resistance: drug efflux, melanoma, vemurafenib, lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens, lcsh:RC254-282, Article, new therapeutic lead, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

Melanoma patients harboring the BRAFV600E mutation are treated with vemurafenib. Almost all of them ultimately acquire resistance, leading to disease progression. Here, we find that a small molecule from a marine sponge, panicein A hydroquinone (PAH), overcomes resistance of BRAFV600E melanoma cells to vemurafenib, leading to tumor elimination in corresponding human xenograft models in mice. We report the synthesis of PAH and demonstrate that this compound inhibits the drug efflux activity of the Hedgehog receptor, Patched. Our SAR study allowed identifying a key pharmacophore responsible for this activity. We showed that Patched is strongly expressed in metastatic samples from a cohort of melanoma patients and is correlated with decreased overall survival. Patched is a multidrug transporter that uses the proton motive force to efflux drugs. This makes its function specific to cancer cells, thereby avoiding toxicity issues that are commonly observed with inhibitors of ABC multidrug transporters. Our data provide strong evidence that PAH is a highly promising lead for the treatment of vemurafenib resistant BRAFV600E melanoma.

Published
2020
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28. Role of the unique, non-essential phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt) in Corynebacterium glutamicum

Author

Christiane Dietrich, Laila Sago, David Cornu, Célia de Sousa d’Auria, Niloofar Mohiman, Muriel Masi, Mohamed Chami, Cécile Labarre, Christophe Salmeron, Manuela Argentini, Nicolas Bayan, Christine Houssin, Nathalie Dautin, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), Institut de la Vision, Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Biologie Moléculaire des Corynébactéries et Mycobactéries (CORYNE), Département Microbiologie (Dpt Microbio), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Institut de Biologie Intégrative de la Cellule (I2BC), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Paris-Saclay-Centre National de la Recherche Scientifique (CNRS), SICaPS (SICaPS), Département Plateforme (PF I2BC), Center for Cellular Imaging and NanoAnalytics, Biozentrum [Basel, Suisse], University of Basel (Unibas)-University of Basel (Unibas), Enveloppes Bactériennes et Antibiotiques (ENVBAC), Observatoire océanologique de Banyuls (OOB), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Dautin, Nathalie, Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Phosphatidylglycerol, Signal peptide, 0303 health sciences, Glycosylation, cell envelope, 030306 microbiology, phosphatidylglycerol::prolipoprotein diacylglyceryl transferase, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, [SDV.BBM.BM] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Signal peptidase II, Translocon, Microbiology, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Corynebacterium glutamicum, lipoproteins, 03 medical and health sciences, chemistry.chemical_compound, Secretory protein, chemistry, Biochemistry, lipids (amino acids, peptides, and proteins), [SDV.MP.BAC] Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Cell envelope, 030304 developmental biology
Abstract

Bacterial lipoproteins are secreted proteins that are post-translationally lipidated. Following synthesis, preprolipoproteins are transported through the cytoplasmic membrane via the Sec or Tat translocon. As they exit the transport machinery, they are recognized by a phosphatidylglycerol::prolipoprotein diacylglyceryl transferase (Lgt), which converts them to prolipoproteins by adding a diacylglyceryl group to the sulfhydryl side chain of the invariant Cys+1residue. Lipoprotein signal peptidase (LspA or signal peptidase II) subsequently cleaves the signal peptide, liberating the α-amino group of Cys+1, which can eventually be further modified. Here, we identified thelgtandlspAgenes fromCorynebacterium glutamicumand found that they are unique but not essential. We found that Lgt is necessary for the acylation and membrane anchoring of two model lipoproteins expressed in this species: MusE, aC. glutamicummaltose-binding lipoprotein, and LppX, aMycobacterium tuberculosislipoprotein. However, Lgt is not required for these proteins’ signal peptide cleavage, or for LppX glycosylation. Taken together, these data show that inC. glutamicumthe association of some lipoproteins with membranes through the covalent attachment of a lipid moiety is not essential for further post-translational modification.

Published
2020
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29. Biophysical characterisation of the novel zinc binding property in Suppressor of Fused

Author

Jabrani, Amira, Makamte, Staëlle, Moreau, Émilie, Gharbi, Yasmine, Plessis, Anne, Bruzzone, Lucia, Sanial, Matthieu, Biou, Valérie, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Laboratoire d'Enzymologie et Biochimie Structurales (LEBS), Centre National de la Recherche Scientifique (CNRS), Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
[SDV]Life Sciences [q-bio], lcsh:R, lcsh:Medicine, lcsh:Q, lcsh:Science, ComputingMilieux_MISCELLANEOUS, Article
Abstract

Suppressor of Fused (SUFU) is a highly conserved protein that acts as a negative regulator of the Hedgehog (HH) signalling pathway, a major determinant of cell differentiation and proliferation. Therefore, SUFU deletion in mammals has devastating effects on embryo development. SUFU is part of a multi-protein cytoplasmic signal-transducing complex. Its partners include the Gli family of transcription factors that function either as repressors, or as transcription activators according to the HH activation state. The crystal structure of SUFU revealed a two-domain arrangement, which undergoes a closing movement upon binding a peptide from Gli1. There remains however, much to be discovered about SUFU’s behaviour. To this end, we expressed recombinant, full-length SUFU from Drosophila, Zebrafish and Human. Guided by a sequence analysis that revealed a conserved potential metal binding site, we discovered that SUFU binds zinc. This binding was found to occur with a nanomolar affinity to SUFU from all three species. Mutation of one histidine from the conserved motif induces a moderate decrease in affinity for zinc, while circular dichroism indicates that the mutant remains structured. Our results reveal new metal binding affinity characteristics about SUFU that could be of importance for its regulatory function in HH.

Published
2017
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30. Bacterial ABC transporters of iron containing compounds

Author

Philippe Delepelaire, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Siderophore, siderophore, Heme binding, Iron, substrate-binding protein, Siderophores, ATP-binding cassette transporter, Biology, Microbiology, 03 medical and health sciences, chemistry.chemical_compound, Heme-Binding Proteins, heme, Molecular Biology, Heme, 030304 developmental biology, 0303 health sciences, Bacteria, 030306 microbiology, Binding protein, Cell Membrane, Transporter, Biological Transport, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, General Medicine, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Transmembrane domain, chemistry, Biochemistry, Cytoplasm, ATP-Binding Cassette Transporters, Iron Compounds
Abstract

International audience; Iron acquisition is an essential aspect of cell physiology for most bacteria. Although much is known about how bacteria initially recognize the various iron sources they can encounter, whether siderophore, heme, host iron/heme binding proteins, much less is known about how the iron containing compounds (Fe 2+ , Fe 3+ , Fe 3+-siderophore complex or heme) are transported across the cytoplasmic membrane. This last transport step is powered by specific ABC (ATP-Binding-Cassette) transporters, made up of a substrate binding protein (SBP) that delivers its cargo to the TMD (TransMembrane Domain) of the ABC transporter triggering the entry of the substrate inside the cytoplasm upon catalytic activity of the ABC module. This review focuses on structural aspects of the functioning of such ABC transporters with the most part devoted to the substrate binding proteins.

Published
2019
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31. Improved protection against Chlamydia muridarum using the native major outer membrane protein trapped in Resiquimod-carrying amphipols and effects in protection with addition of a Th1 (CpG-1826) and a Th2 (Montanide ISA 720) adjuvant

Author

Delia F. Tifrea, Melanie J. Cocco, Luis M. de la Maza, Christel Le Bon, Manuela Zoonens, Sukumar Pal, zoonens, manuela, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Public Health Service grant AI067888 and AI092129 from the National Institute of Allergy and Infectious Diseases.The Centre National de la Recherche Scientifique (CNRS), the Université de Paris, and the 'Initiative d’Excellence' program from the French State (Grant 'DYNAMO', ANR-11-LABX-0011-01).

Subjects
Chlamydia muridarum, Resiquimod, [SDV]Life Sciences [q-bio], medicine.medical_treatment, TLR7/8 adjuvants, 030231 tropical medicine, amphipols, Article, 03 medical and health sciences, chemistry.chemical_compound, Mice, 0302 clinical medicine, Immune system, Adjuvants, Immunologic, vaccine, medicine, Animals, 030212 general & internal medicine, Mice, Inbred BALB C, General Veterinary, General Immunology and Microbiology, biology, Chemistry, major outer membrane protein, Public Health, Environmental and Occupational Health, Imidazoles, TLR9, A8 35, Chlamydia Infections, biology.organism_classification, Antibodies, Bacterial, In vitro, 3. Good health, [SDV] Life Sciences [q-bio], Infectious Diseases, Oligodeoxyribonucleotides, Immunology, Bacterial Vaccines, biology.protein, Molecular Medicine, Nasal administration, Antibody, Adjuvant, Bacterial Outer Membrane Proteins
Abstract

International audience; A new vaccine formulated with the Chlamydia muridarum native major outer membrane protein (nMOMP) and amphipols was assessed in an intranasal (i.n.) challenge mouse model. nMOMP was trapped either in amphipol A8-35 (nMOMP/A8-35) or in A8-35 conjugated with Resiquimod (nMOMP/Resiq-A8-35), a TLR7/8 agonist added as adjuvant. The effects of free Resiquimod and/or additional adjuvants, Montanide ISA 720 (TLR independent) and CpG-1826 (TLR9 agonist), were also evaluated. Immunization with nMOMP/A8-35 alone administered i.n. was used as negative adjuvant-control group, whereas immunizations with C. muridarum elementary bodies (EBs) and MEM buffer, administered i.n., were used as positive and negative controls, respectively. Vaccinated mice were challenged i.n. with C. muridarum and changes in body weight, lungs weight and recovery of Chlamydia from the lungs were evaluated. All the experimental groups showed protection when compared with the negative control group. Resiquimod alone produced weak humoral and cellular immune responses, but both Montanide and CpG-1826 showed significant increases in both responses. The addition of CpG-1826 alone switched immune responses to be Th1-biased. The most robust protection was elicited in mice immunized with the three adjuvants and conjugated Resiquimod. Increased protection induced by the Resiquimod covalently linked to A8-35, in the presence of Montanide and CpG-1826 was established based on a set of parameters: 1) the ability of the antibodies to neutralize C. muridarum, 2) the increased proliferation of T-cells in vitro accompanied by higher production of IFN-, IL-6 and IL-17, 3) the decreased body weight loss over the 10 days after challenge, and 4) the number of IFUs recovered from the lungs at day 10 post challenge. In conclusion, a vaccine formulated with the C. muridarum nMOMP bound to amphipols conjugated with Resiquimod enhances protective immune responses that can be further improved by the addition of Montanide and CpG-1826.

Published
2019

32. BAmSA: Visualising transmembrane regions in protein complexes using biotinylated amphipols and electron microscopy

Author

Fabrice Giusti, Hager Souabni, Rémi Fronzes, Manuela Zoonens, Chiara Rapisarda, Jean-Luc Popot, Francesca Gubellini, Thomas Noe Perry, Biologie Structurale de la Sécrétion Bactérienne, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Européen de Chimie et Biologie (IECB), Université de Bordeaux (UB)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), This work was supported by the Centre National de la Recherche Scientifique (CNRS), the University Paris-7 (Université Sorbonne Paris Cité), the 'Initiative d'Excellence' program from the French State (Grant 'DYNAMO', ANR-11-LABX-0011-01) and by the Institut Pasteur (Paris, France)., We are grateful to E.A. Berry and L.-S. Huang (University of Syracuse, Syracuse, NY) for their generous gift of purified cytochrome bc1. We wish to thank Dr. Han Remaut (VIB-VUB Center for Structural Biology, Brussel) for the kind gift of CsgG plasmid and advices on CsgG. We also thank Sandrine Masscheleyn for masse spectrometry analysis of mSA samples., ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Laboratoire de Chimie Physique D'Orsay (LCPO), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), European Institute of Chemistry and Biology, Université Sciences et Technologies - Bordeaux 1, Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), and Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, Polymers, 030310 physiology, MESH: Membrane Proteins/ultrastructure, Biochemistry, Negative Staining, law.invention, chemistry.chemical_compound, Electron Transport Complex III, law, Labelling, Membrane proteins, MESH: Animals, MESH: Negative Staining, ComputingMilieux_MISCELLANEOUS, Tagged amphipols, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Chemistry, Escherichia coli Proteins, Resolution (electron density), MESH: Streptavidin/chemistry, Transmembrane protein, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Cattle, MESH: Microscopy, Electron, Biotinylation, MESH: Lipoproteins/metabolism, MESH: Models, Molecular, Streptavidin, Lipoproteins, Biophysics, MESH: Polymers/chemistry, Context (language use), MESH: Escherichia coli Proteins/metabolism, 03 medical and health sciences, Animals, MESH: Biotinylation, MESH: Electron Transport Complex III/metabolism, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], 030304 developmental biology, Cell Membrane, Cell Biology, MESH: Cell Membrane/ultrastructure, Microscopy, Electron, [CHIM.POLY]Chemical Sciences/Polymers, Membrane protein, Cattle, Electron microscope, Negative stain electron microscopy
Abstract

International audience; Membrane protein (MP) complexes play key roles in all living cells. Their structural characterisation is hampered by difficulties in purifying and crystallising them. Recent progress in electron microscopy (EM) have revolutionised the field, not only by providing higher-resolution structures for previously characterised MPs but also by yielding first glimpses into the structure of larger and more challenging complexes, such as bacterial secretion systems. However, the resolution of pioneering EM structures may be difficult and their interpretation requires clues regarding the overall organisation of the complexes. In this context, we present BAmSA, a new method for localising transmembrane (TM) regions in MP complexes, using a general procedure that allows tagging them without resorting to neither genetic nor chemical modification. Labels bound to TM regions can be visualised directly on raw negative-stain EM images, on class averages, or on three-dimensional reconstructions, providing a novel strategy to explore the organisation of MP complexes.

Published
2019
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33. Bacteria‐Based Production of Thiol‐Clickable, Genetically Encoded Lipid Nanovesicles

Author

Jorge Royes, Oana Ilioaia, Quentin Lubart, Federica Angius, Galina V. Dubacheva, Marta Bally, Bruno Miroux, Christophe Tribet, Processus d'Activation Sélective par Transfert d'Energie Uni-électronique ou Radiatif (UMR 8640) (PASTEUR), Département de Chimie - ENS Paris, École normale supérieure - Paris (ENS Paris)-École normale supérieure - Paris (ENS Paris)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Laboratoire des matériaux et du génie physique (LMGP), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique de Grenoble (INPG), Laboratoire de Photophysique et Photochimie Supramoléculaires et Macromoléculaires (PPSM), École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS), Chalmers University of Technology [Göteborg], École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-École normale supérieure - Paris (ENS Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), École normale supérieure - Cachan (ENS Cachan)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), and ANR-17-CE09-0007,GenCaps,Bioproduction de capsules fonctionnelles génétiquement encodées(2017)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, 010405 organic chemistry, [CHIM]Chemical Sciences, General Medicine, 010402 general chemistry, 01 natural sciences, 0104 chemical sciences
Abstract

International audience; Despite growing research efforts on the preparation of (bio)functional liposomes, synthetic capsules cannot reach the densities of protein loading and the control over peptide display that is achieved by natural vesicles. Here we present a microbial platform for high yield production of lipidic nanovesicles, with clickable thiol moieties in their outer corona. These nanovesicles show low size dispersity, are decorated with a dense, perfectly oriented and customizable corona of transmembrane polypeptides. In addition, this approach enables encapsulation of soluble proteins into the nanovesicles. Due to the mild preparation and loading conditions (absence of organic solvents, pH gradients or detergents) and their straightforward surface functionalization taking advantage of the diversity of commercially-available maleimide derivatives, engineering bacterial-based proteoliposomes are an attractive eco-friendly alternative that can outperform current liposome preparation methods.

Published
2019
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34. Uncoupling protein 2 protects mice from aging

Author

Bruno Miroux, Markus Schwaninger, Johannes Mayer, Saleh M. Ibrahim, Karin Scharffetter-Kochanek, Misa Hirose, Falko Lange, Paul Schilf, Olaf Jöhren, Rüdiger Köhling, Gesine Reichart, Christian Sina, Christian D. Sadik, Pallab Maity, Institute for Experimental and Clinical Pharmacology and Toxicology [Lübeck, Germany], University of Lübeck [Germany], Department of Dermatology and Allergic Diseases, Universität Ulm - Ulm University [Ulm, Allemagne], University of Rostock, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), and Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP)

Subjects
Male, Serum, 0301 basic medicine, Premature aging, Aging, medicine.medical_specialty, [SDV]Life Sciences [q-bio], Transgene, medicine.medical_treatment, Biology, Mice, 03 medical and health sciences, Internal medicine, medicine, Animals, Uncoupling protein, Uncoupling Protein 2, Longitudinal Studies, Insulin-Like Growth Factor I, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Mice, Knockout, Insulin, Lipid metabolism, Cell Biology, Accelerated aging, Phenotype, Transport protein, Cross-Sectional Studies, 030104 developmental biology, Endocrinology, Molecular Medicine, Female
Abstract

Uncoupling protein (UCP) 2 is a mitochondrial transporter protein that plays various roles in cellular metabolism, including the glucose and lipid metabolism. Polymorphisms in UCP2 are associated with longevity in humans. In line with this, mice carrying the UCP2 transgene under the control of hypocretin promoter were reported to have an extended lifespan, while, conversely, mice deficient in Ucp2 demonstrated a significantly shorter lifespan. In this study, we examined the phenotype of aging in a large colony of Ucp2-deficient (Ucp2(-/-)) mice on the molecular level. We have found that the significantly shorter lives of Ucp2(-/-) mice is the result of an accelerated aging process throughout their entire lifespan. Thus, Ucp2(-/-) mice not only earlier gained sexual maturity, but also earlier progressed into an aging phenotype, reflected by a decrease in body weight, increased neutrophil numbers, and earlier emergence of spontaneous ulcerative dermatitis. Intriguingly, on the molecular level this acceleration in aging predominantly driven by increased levels of circulating IGF-1 in Ucp2(-/-) mice, hinting at a crosstalk between UCP2 and the classical Insulin/IGF-1 signaling aging pathway.

Published
2016
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35. NMR analysis of GPCR conformational landscapes and dynamics

Author

Laurent Catoire, Karine Moncoq, Elodie Point, Jean-Louis Banères, Alexandre Pozza, Marina Casiraghi, Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Stanford University, Institut de biologie physico-chimique (IBPC (FR_550)), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)

Subjects
0301 basic medicine, Models, Molecular, Magnetic Resonance Spectroscopy, Protein Conformation, Allosteric regulation, 030209 endocrinology & metabolism, Context (language use), Computational biology, Crystallography, X-Ray, Biochemistry, Receptors, G-Protein-Coupled, 03 medical and health sciences, 0302 clinical medicine, Endocrinology, Molecular level, Animals, Humans, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Molecular Biology, ComputingMilieux_MISCELLANEOUS, G protein-coupled receptor, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Mechanism (biology), Cryoelectron Microscopy, Energy landscape, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 030104 developmental biology, Structural biology, Function (biology), Protein Binding, Signal Transduction
Abstract

Understanding the signal transduction mechanism mediated by the G Protein-Coupled Receptors (GPCRs) in eukaryote cells represents one of the main issues in modern biology. At the molecular level, various biophysical approaches have provided important insights on the functional plasticity of these complex allosteric machines. In this context, X-ray crystal structures published during the last decade represent a major breakthrough in GPCR structural biology, delivering important information on the activation process of these receptors through the description of the three-dimensional organization of their active and inactive states. In complement to crystals and cryo-electronic microscopy structures, information on the probability of existence of different GPCR conformations and the dynamic barriers separating those structural sub-states is required to better understand GPCR function. Among the panel of techniques available, nuclear magnetic resonance (NMR) spectroscopy represents a powerful tool to characterize both conformational landscapes and dynamics. Here, we will outline the potential of NMR to address such biological questions, and we will illustrate the functional insights that NMR has brought in the field of GPCRs in the recent years.

Published
2018
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36. Self-organized wave-like beating of actin bundles in a minimal actomyosin system

Author

Pochitaloff-Huvalé, Marie, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Sorbonne Université, Pascal Martin, Mathilde Badoual, Institut de biologie physico-chimique (IBPC), and STAR, ABES

Subjects
Self-organization, Spontaneous oscillations, Auto-organisation, Myosin, Oscillations spontanées, macromolecular substances, [PHYS.PHYS.PHYS-CHEM-PH] Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], In vitro, Cytosquelette, Myosine, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Actine, Cytoskeleton, Actin
Abstract

The emergent active behaviors of molecular motors assemblies and cytoskeletal filaments systems remain poorly understood, though individual molecules have been extensively characterized. By controlling the geometry of actin polymerization with surface micropatterns of a nucleation promoting factor, we were able to demonstrate in vitro the emergence of flagellar-like beating of bundles of parallel actin filaments in the presence of myosin motors. We worked with both myosin V and heavy-meromyosin II. The waveform of oscillation was similar for the two types of motors, but oscillations with myosin II were one order of magnitude faster than with myosin Va. In both cases, a bending wave traveled at a uniform speed from the anchored base of the actin bundle towards the tip. As polymerization occurred, the actin bundle elongated at a constant speed, resulting in an increase of the oscillation period, but the speed of the traveling bending wave remains constant. GFP-tagged myosin V revealed the presence of a myosin concentration peak within the actin bundle. Strikingly, myosin V motors were locally recruited within the actin bundle, before a concentration wave propagated towards the bundle’s tip in concert with the actin bending wave. These results revealed a novel form of coupling between the myosin affinity for actin and the actin bundle shape. Our work demonstrates that active flagellar-like beating emerges as an intrinsic property of polar bundles of filaments in interaction with molecular motors. Structural control over the self-assembly process provides key information to clarify the underlying physical principles of flagellar-like beating., L’interaction d’assemblées de moteurs et de filaments du cytosquelette donne naissance à des comportements actifs qui demeurent peu compris, malgré la large caractérisation de leurs molécules individuellement. En contrôlant la géométrie de polymérisation de l’actine via des micropatrons surfaciques de nucléation, nous avons observé in vitro l’émergence de battements de faisceaux de filaments parallèles d’actine en présence de myosines en solution (myosine V ou HMM II (Heavy MeroMyosine II)). La forme du battement est similaire pour les deux types de moteurs, mais avec des oscillations un ordre de grandeur plus rapides avec la myosine II qu’avec la myosine V. Dans les deux cas, une onde de déformation transverse se propage à vitesse uniforme de la base à la pointe du faisceau d’actine. Avec la polymérisation, les faisceaux d’actine s’allongent à vitesse constante : la période croît, mais la vitesse de l’onde mécanique reste inchangée. L’utilisation de myosines-GFP a révélé un pic de concentration et un recrutement localisé des myosines au sein du faisceau d’actine, avant qu’une onde de concentration ne se propage vers la pointe de concert avec l’onde mécanique de l’actine. Ces résultats présentent une nouvelle forme de couplage entre l’affinité des myosines à l’actine et la forme du faisceau d’actine. Ce travail de thèse décrit l’émergence de battements actifs imitant ceux des flagelles comme une propriété intrinsèque de l’interaction de filaments polaires et de moteurs moléculaires. Le contrôle de la structure lors du processus d’auto-organisation fournit des informations clés pour étudier les principes physiques génériques du battement flagellaire.

Published
2018

37. Specific cardiolipin–SecY interactions are required for proton-motive force stimulation of protein secretion

Author

Bruno Miroux, Ian Collinson, Robin A. Corey, Marina Casiraghi, Euan Pyle, William J. Allen, Daniel W. Watkins, Ignacio Arechaga, Argyris Politis, Biotechnology and Biological Sciences Research Council (UK), Wellcome Trust, Centre National de la Recherche Scientifique (France), Imperial College London, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Department of Biochemistry, School of Medical Sciences, ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), ANR-17-CE09-0007,GenCaps,Bioproduction de capsules fonctionnelles génétiquement encodées(2017), and Universidad de Cantabria

Subjects
0301 basic medicine, SecYEG, Membrane lipids, Molecular Dynamics Simulation, Biology, Molecular dynamics, Motor protein, 03 medical and health sciences, chemistry.chemical_compound, Native mass spectrometry, ATP hydrolysis, Escherichia coli, Cardiolipin, Thermotoga maritima, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Bacterial Secretion Systems, ComputingMilieux_MISCELLANEOUS, Protein translocation, Multidisciplinary, Chemiosmosis, Escherichia coli Proteins, Proton-Motive Force, Biological Sciences, biology.organism_classification, Transmembrane protein, Transport protein, 030104 developmental biology, chemistry, Biochemistry, Biophysics, SEC Translocation Channels
Abstract

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions., This work was funded by the Biotechnology and Biological Sciences Research Council (Grants BB/M003604/1, BB/I008675/1, and BB/N015126/1) and the Wellcome Trust (Grants 104632 and 109854/Z/15/Z). The mass spectrometry work was supported by the Centre National de la Recherche Scientifique, INSERM (Grant “DYNAMO,” ANR-11-LABEX-0011-01 to M.C.) and the Région Ile de France for cofunding the Le Service d’Analyse des Médicaments et Métabolites Mass Spectrometry Facility at IPSIT. E.P. is the recipient of an Imperial College London Institute of Chemical Biology Engineering and Physical Sciences Research Council (EPSRC) Centre for Doctoral Training studentship.

Published
2018
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38. Overall Structures of Mycobacterium tuberculosis DNA Gyrase Reveal the Role of a Corynebacteriales GyrB-Specific Insert in ATPase Activity

Author

Claudine Mayer, Pedro M. Alzari, Clément Giffard, Aurélien Thureau, Stéphanie Petrella, Françoise Bonneté, Alexandra Aubry, Estelle Capton, Bertrand Raynal, CCSD, Accord Elsevier, Microbiologie structurale - Structural Microbiology (Microb. Struc. (UMR_3528 / U-Pasteur_5)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et des Maladies Infectieuses (CIMI), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Biophysique Moléculaire (Plate-forme), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), This work was funded by Institut Pasteur, Université Paris Diderot, and Sorbonne-Université (France)., Université Paris Diderot - Paris 7 (UPD7)-Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre d'Immunologie et de Maladies Infectieuses (CIMI), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)

Subjects
type IIA topoisomerases, Models, Molecular, Protein Conformation, alpha-Helical, ATPase, Gene Expression, Crystallography, X-Ray, ATPase activity, DNA gyrase, Adenosine Triphosphate, Structural Biology, Cloning, Molecular, Adenosine Triphosphatases, molecular machine, 0303 health sciences, biology, Chemistry, Drug discovery, 030302 biochemistry & molecular biology, Molecular machine, Recombinant Proteins, 3. Good health, Biochemistry, DNA Gyrase, Protein Binding, Adenylyl Imidodiphosphate, Genetic Vectors, DNA-binding protein, Corynebacterium, fluoroquinolone, Cleavage (embryo), [PHYS.PHYS.PHYS-CHEM-PH] Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Mycobacterium tuberculosis, Corynebacteriales, 03 medical and health sciences, Escherichia coli, Atpase activity, Protein Interaction Domains and Motifs, Amino Acid Sequence, Protein Structure, Quaternary, SAXS experiments, Molecular Biology, 030304 developmental biology, Binding Sites, Sequence Homology, Amino Acid, DNA, biology.organism_classification, Kinetics, biology.protein, Protein Conformation, beta-Strand, X-ray structure, [PHYS.PHYS.PHYS-CHEM-PH]Physics [physics]/Physics [physics]/Chemical Physics [physics.chem-ph], Apoproteins, Sequence Alignment
Abstract

International audience; Despite sharing common features, previous studies have shown that gyrases from different species have been modified throughout evolution to modulate their properties. Here, we report two crystal structures of Mycobacterium tuberculosis DNA gyrase, an apo and AMPPNP-bound form at 2.6-Å and 3.3-Å resolution, respectively. These structures provide high-resolution structural data on the quaternary organization and interdomain connections of a gyrase (full-length GyrB-GyrA57)2 thus providing crucial inputs on this essential drug target. Together with small-angle X-ray scattering studies, they revealed an "extremely open" N-gate state, which persists even in the DNA-free gyrase-AMPPNP complex and an unexpected connection between the ATPase and cleavage core domains mediated by two Corynebacteriales-specific motifs, respectively the C-loop and DEEE-loop. We show that the C-loop participates in the stabilization of this open conformation, explaining why this gyrase has a lower ATPase activity. Our results image a conformational state which might be targeted for drug discovery.

Published
2018
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39. Perturbations of Native Membrane Protein Structure in Alkyl Phosphocholine Detergents: A Critical Assessment of NMR and Biophysical Studies

Author

Timothy A. Cross, Laurent Catoire, Eva Pebay-Peyroula, Bruno Miroux, Paul Schanda, Gianluigi Veglia, Christophe Chipot, Edmund R.S. Kunji, Nicole Zitzmann, François Dehez, Jason R. Schnell, Structure et Réactivité des Systèmes Moléculaires Complexes (SRSMC), Institut de Chimie du CNRS (INC)-Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Physique et Chimie Théoriques (LPCT), Department of Biochemistry [Oxford], University of Oxford, 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), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie et chimie des protéines [Lyon] (IBCP), Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS), Medical Research Council Mitochondrial Biology Unit, University of Cambridge [UK] (CAM), Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, University of Minnesota [Twin Cities] (UMN), University of Minnesota System-University of Minnesota System-Department of Biochemistry, Molecular Biology and Biophysics, National High Magnetic Field Laboratory (NHMFL), Florida State University [Tallahassee] (FSU)-NSF, Chipot, Christophe [0000-0002-9122-1698], Zitzmann, Nicole [0000-0003-1969-4949], Schanda, Paul [0000-0002-9350-7606], Apollo - University of Cambridge Repository, Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS), University of Oxford [Oxford], 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), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), University of Minnesota [Twin Cities], Université de Lorraine (UL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), 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
0301 basic medicine, Models, Molecular, Protein Folding, Magnetic Resonance Spectroscopy, Protein Conformation, Phosphorylcholine, Detergents, Cellular functions, Review, 010402 general chemistry, 01 natural sciences, Biophysical Phenomena, 03 medical and health sciences, chemistry.chemical_compound, Animals, Humans, Alkyl, Micelles, Phosphocholine, chemistry.chemical_classification, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Protein Stability, Cell Membrane, Membrane protein solubilization, Membrane Proteins, Biological membrane, General Chemistry, 0104 chemical sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Kinetics, 030104 developmental biology, chemistry, Membrane protein, Solubility, Biophysics, Critical assessment, Hydrophobic and Hydrophilic Interactions
Abstract

International audience; Membrane proteins perform a host of vital cellular functions. Deciphering the molecular mechanisms whereby they fulfill these functions requires detailed biophysical and structural investigations. Detergents have proven pivotal to extract the protein from its native surroundings. Yet, they provide a milieu that departs significantly from that of the biological membrane, to the extent that the structure, the dynamics, and the interactions of membrane proteins in detergents may considerably vary, as compared to the native environment. Understanding the impact of detergents on membrane proteins is, therefore, crucial to assess the biological relevance of results obtained in detergents. Here, we review the strengths and weaknesses of alkyl phosphocholines (or foscholines), the most widely used detergent in solution-NMR studies of membrane proteins. While this class of detergents is often successful for membrane protein solubilization, a growing list of examples points to destabilizing and denaturing properties, in particular for α-helical membrane proteins. Our comprehensive analysis stresses the importance of stringent controls when working with this class of detergents and when analyzing the structure and dynamics of membrane proteins in alkyl phosphocholine detergents.

Published
2018
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40. Microbial expression systems for membrane proteins

Author

Marvin V. Dilworth, Roslyn M. Bill, David R. Poyner, Stephane R. Gross, Peter J. F. Henderson, Bruno Miroux, Pikyee Ma, Karine Moncoq, Kim E. Bettaney, Mathilde S. Piel, Ji Luo, David Sharples, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Bettaney, K, Ma, P, Luo, J, Sharples, D, Poyner, D, Gross, S, Moncoq, K, Miroux, B, and Bill, RM

Subjects
0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Bacteria, Chemistry, Membrane Proteins, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Promoter, Computational biology, General Biochemistry, Genetics and Molecular Biology, Recombinant Proteins, law.invention, 03 medical and health sciences, 030104 developmental biology, Membrane protein, Cell culture, law, Solubilization, Yeasts, Recombinant DNA, Promoter Regions, Genetic, Molecular Biology, ComputingMilieux_MISCELLANEOUS, Plasmids
Abstract

Despite many high-profile successes, recombinant membrane protein production remains a technical challenge; it is still the case that many fewer membrane protein structures have been published than those of soluble proteins. However, progress is being made because empirical methods have been developed to produce the required quantity and quality of these challenging targets. This review focuses on the microbial expression systems that are a key source of recombinant prokaryotic and eukaryotic membrane proteins for structural studies. We provide an overview of the host strains, tags and promoters that, in our experience, are most likely to yield protein suitable for structural and functional characterization. We also catalogue the detergents used for solubilization and crystallization studies of these proteins. Here, we emphasize a combination of practical methods, not necessarily high-throughput, which can be implemented in any laboratory equipped for recombinant DNA technology and microbial cell culture.

Published
2018
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41. Identification of lipid and saccharide constituents of whole microalgal cells by 13C solid-state NMR

Author

Isabelle Marcotte, Francesca Zito, Réjean Tremblay, Dror E. Warschawski, Bertrand Genard, Alexandre A. Arnold, EADS Innovation Works [Toulouse], EADS - European Aeronautic Defense and Space, Institut des Sciences de la MER de Rimouski (ISMER), Université du Québec à Rimouski (UQAR), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Département de Chimie [Montréal], and Université du Québec à Montréal = University of Québec in Montréal (UQAM)

Subjects
Dynamic filter, [SDV]Life Sciences [q-bio], Microorganism, Biophysics, Storage, Chlamydomonas reinhardtii, Biology, 010402 general chemistry, Cell surface, 01 natural sciences, Biochemistry, Cell wall, 03 medical and health sciences, chemistry.chemical_compound, Organelle, Botany, [CHIM]Chemical Sciences, Cellulose, Picoplankton, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, chemistry.chemical_classification, 0303 health sciences, Membrane, Cell Biology, biology.organism_classification, 6. Clean water, 0104 chemical sciences, chemistry, [SDE]Environmental Sciences, Phytoplankton, Glycoprotein
Abstract

Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater (Chlamydomonas reinhardtii) and marine (Pavlova lutheri and Nannochloropsis oculata) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by 13C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as 13C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR.This article is part of a Special Issue entitled: NMR Spectroscopy for Atomistic Views of Biomembranes and Cell Surfaces. Guest Editors: Lynette Cegelski and David P. Weliky.

Published
2015
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42. Cardiolipin plays an essential role in the formation of intracellular membranes in Escherichia coli

Author

Gerardo Carranza, Bruno Miroux, Audrey Solgadi, Federica Angius, Oana Ilioaia, Ignacio Arechaga, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), Institut Paris-Saclay d’Innovation Thérapeutique (IPSIT), Universidad de Cantabria and Instituto de Biomedicina y Biotecnología de Cantabria (IBBTEC), Departamento de Biología Molecular, ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), Ministerio de Economía y Competitividad (España), Centre National de la Recherche Scientifique (France), Agence Nationale de la Recherche (France), Instituto de Investigación Marqués de Valdecilla, and Région Ile-de-France

Subjects
0301 basic medicine, Cardiolipins, [SDV]Life Sciences [q-bio], Biophysics, Gene Expression, Transferases (Other Substituted Phosphate Groups), F1Fo-ATPase, medicine.disease_cause, Endoplasmic Reticulum, Biochemistry, Time-Lapse Imaging, Fluorescence, 03 medical and health sciences, chemistry.chemical_compound, Bacterial Proteins, medicine, Cardiolipin, Escherichia coli, Electron microscopy, Flow cytometry, Membrane biogenesis, ComputingMilieux_MISCELLANEOUS, Fluorescent Dyes, 030102 biochemistry & molecular biology, ATP synthase, biology, Bacteria, Membrane Proteins, Cell Biology, 3. Good health, Cell biology, Mitochondria, Isoenzymes, Phospholipid, 030104 developmental biology, Membrane, chemistry, Membrane protein, Cytoplasm, Bacterial Proton-Translocating ATPases, biology.protein, Photosynthetic bacteria, Gene Deletion
Abstract

Mitochondria, chloroplasts and photosynthetic bacteria are characterized by the presence of complex and intricate membrane systems. In contrast, non-photosynthetic bacteria lack membrane structures within their cytoplasm. However, large scale over-production of some membrane proteins, such as the fumarate reductase, the mannitol permease MtlA, the glycerol acyl transferase PlsB, the chemotaxis receptor Tsr or the ATP synthase subunit b, can induce the proliferation of intra cellular membranes (ICMs) in the cytoplasm of Escherichia coli. These ICMs are particularly rich in cardiolipin (CL). Here, we have studied the effect of CL in the generation of these membranous structures. We have deleted the three genes (clsA, clsB and clsC) responsible of CL biosynthesis in E. coli and analysed the effect of these mutations by fluorescent and electron microscopy and by lipid mass spectrometry. We have found that CL is essential in the formation of non-lamellar structures in the cytoplasm of E. coli cells. These results could help to understand the structuration of membranes in E. coli and other membrane organelles, such as mitochondria and ER., This work was supported by the Spanish Ministerio de Economía y Competitividad (MINECO) grant BFU2013-49486-EXP (to I A), by the Centre National de la Recherche Scientifique, INSERM, and by the “Initiative d'Excellence” program from the French State (Grant “DYNAMO”, ANR-11-LABEX-0011-01) (to BM). FA is supported by a DYNAMO PhD fellowship. The authors acknowledge Jorge Mata, Dr. F. Madrazo, Dr. M.T. Berciano and Dr. M. Lafarga at the IDIVAL institute in Santander, Spain, for help with the cross-sections of cells. We also like to acknowledge Région Ile de France for co-funding the SAMM MS Facility at IPSIT.

Published
2017
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43. A stromal region of cytochrome $b_6f$ subunit IV is involved in the activation of the Stt7 kinase in Chlamydomonas

Author

Francesca Zito, Stéphanie Blangy, Xenie Johnson, Gilles Peltier, Jean Alric, Pascaline Auroy, Louis Dumas, Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-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), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Photosynthèse & Environnement (P&E), ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011), ANR-14-CE05-0041,ChloroPaths,Production in vivo et in silico de mutants affectant les voies de la photosynthèse(2014), 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)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bioénergie et Microalgues (EBM), Institut de biologie physico-chimique (IBPC), and ANR-11-LABX-0011/11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011)

Subjects
0106 biological sciences, 0301 basic medicine, Cytochrome, Protein subunit, Plastoquinone, 01 natural sciences, environment and public health, 03 medical and health sciences, chemistry.chemical_compound, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Photosystem, Multidisciplinary, biology, Cytochrome b6f complex, Autophosphorylation, Chlamydomonas, [SDV.BV.BOT]Life Sciences [q-bio]/Vegetal Biology/Botanics, biology.organism_classification, Cell biology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], enzymes and coenzymes (carbohydrates), 030104 developmental biology, chemistry, Protein kinase domain, Biochemistry, embryonic structures, biology.protein, cardiovascular system, 010606 plant biology & botany
Abstract

International audience; The cytochrome (cyt) $b_6f$ complex and Stt7 kinase regulate the antenna sizes of photosystems I and II through state transitions, which are mediated by a reversible phosphorylation of light harvesting complexes II, depending on the redox state of the plastoquinone pool. When the pool is reduced, the cyt $b_6f$ activates the Stt7 kinase through a mechanism that is still poorly understood. After random mutagenesis of the chloroplast petD gene, coding for subunit IV of the cyt $b_6f$ complex, and complementation of a ΔpetDhost strain by chloroplast transformation, we screened for impaired state transitions in vivo by chlorophyll fluorescence imaging. We show that residues Asn122, Tyr124, and Arg125 in the stromal loop linking helices F and G of cyt $b_6f$ subunit IV are crucial for state transitions. In vitro reconstitution experiments with purified cyt $b_6f$ and recombinant Stt7 kinase domain show that cyt $b_6f$ enhances Stt7 autophosphorylation and that the Arg125 residue is directly involved in this process. The peripheral stromal structure of the cyt $b_6f$ complex had, until now, no reported function. Evidence is now provided of a direct interaction with Stt7 on the stromal side of the membrane

Published
2017
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44. Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

Author

Jacqueline W. Chung, Jean-Luc Popot, Lin Yang, James W. Coulton, Aleksandr Sverzhinsky, Isabel Moraes, Marc Allaire, Dewang Ma, Manuela Zoonens, Shuo Qian, Oak Ridge National Laboratory [Oak Ridge] (ORNL), UT-Battelle, LLC, School of Public Health, The University of Hong Kong (HKU), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)

Subjects
Polymers, Protein Conformation, Physiology, [SDV]Life Sciences [q-bio], Size-exclusion chromatography, Biophysics, Plasma protein binding, Surface-Active Agents, Protein structure, Escherichia coli, ComputingMilieux_MISCELLANEOUS, Gel electrophoresis, Binding Sites, Chromatography, Propylamines, Chemistry, Escherichia coli Proteins, Cell Biology, Transmembrane protein, Solubility, Membrane protein, Membrane protein complex, Multiprotein Complexes, Bacterial outer membrane, Hydrophobic and Hydrophilic Interactions, Bacterial Outer Membrane Proteins, Protein Binding
Abstract

Nutrient import across Gram-negative bacte- ria's outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB-ExbD-TonB. Having purified the ExbB4- ExbD2 complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water- soluble membrane protein/amphipol complex. Properties of the ExbB4-ExbD2 complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifuga- tion, respectively. The structural arrangement of ExbB4- ExbD2 was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated am- phipol. The amphipol-trapped ExbB4-ExbD2 complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB6-ExbD4, and propose a structural arrange- ment of its transmembrane a-helical domains.

Published
2014
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45. How Amphipols Embed Membrane Proteins: Global Solvent Accessibility and Interaction with a Flexible Protein Terminus

Author

Jean-Luc Popot, Manuela Zoonens, Sebastian Hiller, Manuel Etzkorn, Laurent J. Catoire, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), École Nationale Supérieure de Techniques Avancées (ENSTA Paris), Centre National de la Recherche Scientifique (CNRS), Biozentrum [Basel, Suisse], and University of Basel (Unibas)

Subjects
Hydrolases, Polymers, Protein Conformation, Physiology, [SDV]Life Sciences [q-bio], Biophysics, Surface-Active Agents, Protein Interaction Mapping, Lipid bilayer, Integral membrane protein, ComputingMilieux_MISCELLANEOUS, Propylamines, biology, Chemistry, Escherichia coli Proteins, Peripheral membrane protein, Bacteriorhodopsin, Cell Biology, Transmembrane protein, Protein Structure, Tertiary, Crystallography, Membrane, Solubility, Membrane protein, Bacteriorhodopsins, Solvents, biology.protein, Bacterial outer membrane, Hydrophobic and Hydrophilic Interactions, Bacterial Outer Membrane Proteins, Protein Binding
Abstract

Amphipathic polymers called amphipols provide a valuable alternative to detergents for keeping integral membrane proteins soluble in aqueous buffers. Here, we characterize spatial contacts of amphipol A8-35 with membrane proteins from two architectural classes: The 8-stranded β-barrel outer membrane protein OmpX and the α-helical protein bacteriorhodopsin. OmpX is well structured in A8-35, with its barrel adopting a fold closely similar to that in dihexanoylphosphocholine micelles. The accessibility of A8-35-trapped OmpX by a water-soluble paramagnetic molecule is highly similar to that in detergent micelles and resembles the accessibility in the natural membrane. For the α-helical protein bacteriorhodopsin, previously shown to keep its fold and function in amphipols, NMR data show that the imidazole protons of a polyhistidine tag at the N-terminus of the protein are exchange protected in the presence of detergent and lipid bilayer nanodiscs, but not in amphipols, indicating the absence of an interaction in the latter case. Overall, A8-35 exhibits protein interaction properties somewhat different from detergents and lipid bilayer nanodiscs, while maintaining the structure of solubilized integral membrane proteins.

Published
2014
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46. From low- to high-potential bioenergetic chains : thermodynamic constraints of Q-cycle function

Author

Felix ten Brink, Frauke Baymann, Wolfgang Nitschke, Lucie Bergdoll, Daniel Picot, David Geffen School of Medicine [Los Angeles], University of California [Los Angeles] (UCLA), University of California-University of California, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines (BIP ), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), University of California (UC)-University of California (UC), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Institut de biologie physico-chimique (IBPC), Laboratoire d'ingénierie des systèmes macromoléculaires (LISM), and Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Cytochrome, [SDV]Life Sciences [q-bio], Biophysics, Photochemistry, Biochemistry, Redox, Q cycle, Cofactor, MESH: Q-cycle, Rieske/cytb complex, Cytochrome bc1 complex, Quinone, Electron bifurcation, Great Oxidation Event, 03 medical and health sciences, chemistry.chemical_compound, Redox titration, Benzoquinones, Electrochemistry, Heme, 030102 biochemistry & molecular biology, biology, Chemiosmosis, Electron Spin Resonance Spectroscopy, Cell Biology, Crystallography, 030104 developmental biology, chemistry, biology.protein, Thermodynamics, Energy Metabolism
Abstract

International audience; The electrochemical parameters of all cofactors in the supercomplex formed by the Rieske/cytb complex and the SoxM/A-type O2-reductase from the menaquinone-containing Firmicute Geobacillus stearothermophilus were determined by spectroelectrochemistry and EPR redox titrations. All redox midpoint potentials (Em) were found to be lower than those of ubi- or plastoquinone-containing systems by a value comparable to the redox potential difference between the respective quinones. In particular, Em values of + 200 mV, − 360 mV, − 220 mV and − 50 mV (at pH 7) were obtained for the Rieske cluster, heme bL, heme bH and heme ci, respectively. Comparable values of − 330 mV, − 200 mV and + 120 mV for hemes bL, bH and the Rieske cluster were determined for an anaerobic Firmicute, Heliobacterium modesticaldum. Thermodynamic constraints, optimization of proton motive force build-up and the necessity of ROS-avoidance imposed by the rise in atmospheric O2 2.5 billion years ago are discussed as putative evolutionary driving forces resulting in the observed redox upshift. The close conservation of the entire redox landscape between low and high potential systems suggests that operation of the Q-cycle requires the precise electrochemical tuning of enzyme cofactors to the quinone substrate as stipulated in P. Mitchell's hypothesis.

Published
2016
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47. Functional Modulation of a G Protein-Coupled Receptor Conformational Landscape in a Lipid Bilayer

Author

Elodie Point, Karine Moncoq, Marina Casiraghi, Ewen Lescop, Jean-Louis Banères, Marjorie Damian, Daniel Lévy, Eric Guittet, Nelly Morellet, Laurent Catoire, Jacky Marie, Physico-chimie moléculaire des membranes biologiques (PCMMB), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Institut de Chimie des Substances Naturelles (ICSN), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC), Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), BioImaging Cell and Tissue Core Facility (PICT-IBiSA), Institut Curie [Paris], Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), Institut de biologie physico-chimique (IBPC), and Institut Curie

Subjects
0301 basic medicine, Agonist, Models, Molecular, medicine.drug_class, G protein, Stereochemistry, Protein Conformation, [SDV]Life Sciences [q-bio], Lipid Bilayers, Receptors, Leukotriene B4, 010402 general chemistry, Ligands, 01 natural sciences, Biochemistry, Catalysis, 03 medical and health sciences, Colloid and Surface Chemistry, Allosteric Regulation, medicine, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Receptor, Lipid bilayer, G protein-coupled receptor, Chemistry, Leukotriene B4 receptor, General Chemistry, 0104 chemical sciences, Coupling (electronics), 030104 developmental biology, Membrane, Biophysics, Signal Transduction
Abstract

International audience; Mapping the conformational landscape of G protein-coupled receptors (GPCRs), and in particular how this landscape is modulated by the membrane environment, is required to gain a clear picture of how signaling proceeds. To this end, we have developed an original strategy based on solution-state nuclear magnetic resonance combined with an efficient isotope labeling scheme. This strategy was applied to a typical GPCR, the leukotriene B4 receptor BLT2, reconstituted in a lipid bilayer. Because of this, we are able to provide direct evidence that BLT2 explores a complex landscape that includes four different conformational states for the unliganded receptor. The relative distribution of the different states is modulated by ligands and the sterol content of the membrane, in parallel with the changes in the ability of the receptor to activate its cognate G protein. This demonstrates a conformational coupling between the agonist and the membrane environment that is likely to be fundamental for GPCR signaling.

Published
2016
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48. Membrane Protein Production in Escherichia coli: Protocols and Rules

Author

Federica Angius, Bruno Miroux, Marc Uzan, Oana Ilioaia, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université de Paris (UP), and ANR-11-LABX-0011,DYNAMO,Dynamique des membranes transductrices d'énergie : biogénèse et organisation supramoléculaire.(2011)

Subjects
0301 basic medicine, 030102 biochemistry & molecular biology, Chemistry, [SDV]Life Sciences [q-bio], [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Protein engineering, medicine.disease_cause, Inclusion bodies, Microbiology, law.invention, 03 medical and health sciences, 030104 developmental biology, Membrane, Biochemistry, Membrane protein, law, medicine, Recombinant DNA, T7 RNA polymerase, Escherichia coli, ComputingMilieux_MISCELLANEOUS, medicine.drug
Abstract

Functional and structural studies on membrane proteins are limited by the difficulty to produce them in large amount and in a functional state. In this review, we provide protocols to achieve high-level expression of membrane proteins in Escherichia coli. The T7 RNA polymerase-based expression system is presented in detail and protocols to assess and improve its efficiency are discussed. Protocols to isolate either membrane or inclusion bodies and to perform an initial qualitative test to assess the solubility of the recombinant protein are also included.

Published
2016
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49. Production of UCP1 a membrane protein from the inner mitochondrial membrane using the cell free expression system in the presence of a fluorinated surfactant

Author

L.-A Barret, Ange Polidori, Manuela Zoonens, Stéphanie Ravaud, Céline Juillan-Binard, Eva Pebay-Peyroula, Bruno Miroux, Iulia Blesneac, Bernard Pucci, 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 des Biomolécules Max Mousseron [Pôle Chimie Balard] (IBMM), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels (LCBOSMV), Avignon Université (AU), Equipe Chimie Bioorganique et Systèmes Amphiphiles, Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC (FR_550)), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-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), Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Montpellier (UM)-Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), 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), Ecole Nationale Supérieure de Chimie de Montpellier (ENSCM)-Université de Montpellier (UM)-Centre National de la Recherche Scientifique (CNRS), and Institut de biologie physico-chimique (IBPC)

Subjects
Hydrocarbons, Fluorinated, Gene Expression, Mitochondrion, Biochemistry, Ion Channels, MESH: Recombinant Proteins, MESH: Mitochondrial Membranes, MESH: Cell-Free System, Cell-free expression, MESH: Animals, Inner mitochondrial membrane, Uncoupling Protein 1, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, 030302 biochemistry & molecular biology, MESH: Surface-Active Agents, MESH: Mitochondrial Proteins, Membrane protein expression, Recombinant Proteins, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], MESH: Cattle, Mitochondrial Membranes, ATP–ADP translocase, Fluorinated surfactants, Vesicle-associated membrane protein 8, MESH: Gene Expression, MESH: Rats, Biophysics, Circular dichroism, Mitochondrial Proteins, Surface-Active Agents, 03 medical and health sciences, Mitochondrial membrane transport protein, Mitochondrial carriers, Uncoupling protein, Escherichia coli, Animals, 030304 developmental biology, Cell-Free System, Cell Biology, Mitochondrial carrier, Rats, MESH: Hydrocarbons, Fluorinated, Membrane protein, Translocase of the inner membrane, MESH: Ion Channels, biology.protein, Cattle
Abstract

International audience; Structural studies of membrane protein are still challenging due to several severe bottlenecks, the first being the overproduction of well-folded proteins. Several expression systems are often explored in parallel to fulfil this task, or alternately prokaryotic analogues are considered. Although, mitochondrial carriers play key roles in several metabolic pathways, only the structure of the ADP/ATP carrier purified from bovine heart mitochondria was determined so far. More generally, characterisations at the molecular level are restricted to ADP/ATP carrier or the uncoupling protein UCP1, another member of the mitochondrial carrier family, which is abundant in brown adipose tissues. Indeed, mitochondrial carriers have no prokaryotic homologues and very few efficient expression systems were described so far for these proteins. We succeeded in producing UCP1 using a cell free expression system based on E. coli extracts, in quantities that are compatible with structural approaches. The protein was synthesised in the presence of a fluorinated surfactant, which maintains the protein in a soluble form. Further biochemical and biophysical analysis such as size exclusion chromatography, circular dichroism and thermal stability, of the purified protein showed that the protein is non-aggregated, monodisperse and well-folded.

Published
2012
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50. Rapid transmembrane diffusion of ceramide and dihydroceramide spin-labelled analogues in the liquid ordered phase

Author

Antje Pohl, Iván López-Montero, Florent Rouvière, Fabrice Giusti, Philippe F. Devaux, Univeridad Compluense de Madrid [Madrid, Spain], Laboratoire de biologie physico-chimique des protéines membranaires (LBPC-PM (UMR_7099)), Institut de biologie physico-chimique (IBPC), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Physico-chimie moléculaire des membranes biologiques (PCMMB), Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7), and Institut de biologie physico-chimique (IBPC (FR_550))

Subjects
Ceramide, Liquid ordered phase, Stereochemistry, Lipid Bilayers, Ceramides, Models, Biological, Phase Transition, Diffusion, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Phosphatidylcholine, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Sphingolipids, 0303 health sciences, [CHIM.ORGA]Chemical Sciences/Organic chemistry, Vesicle, Cell Membrane, Biological membrane, Cell Biology, Sphingolipid, Transmembrane protein, [CHIM.THEO]Chemical Sciences/Theoretical and/or physical chemistry, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Cholesterol, Membrane, chemistry, Phosphatidylcholines, Spin Labels, lipids (amino acids, peptides, and proteins), 030217 neurology & neurosurgery
Abstract

In order to study the basic physical phenomena underlying complex lipid transbilayer movement in biological membranes, we have measured the transmembrane diffusion of spin-labelled analogues of sphingolipids in phosphatidylcholine (PC) large unilamellar vesicles in the absence or presence of cholesterol, going from a fluid ( liquid disordered) l(d), phase to a more viscous, liquid ordered (l(o)), phase. We have found cholesterol to reduce the transverse diffusion of glucosylceramide (GlcCer) and galactosylceramide (GalCer) in a concentration-dependent manner. However, surprisingly, we could neither detect any influence of cholesterol on the rapid flip-flop of ceramide nor on the flip-flop of dihydroceramide, for which the tau(1/2) of flip-flop remains in the order of 1 minute at 20 degrees C in the presence of cholesterol. As a consequence of rapid flip-flop of ceramide in both the l(o) and the l(d) phase, ceramide is likely to distribute between the two monolayers of a membrane, and could in principle partition into segregated domains in each side of the plasma membrane of eukaryotic cells.

Published
2009
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