Publications by authors named "Hugues Nury"

20 Publications

  • Page 1 of 1

Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Nat Methods 2021 01 6;18(1):60-68. Epub 2021 Jan 6.

Structural Biology Brussels, Vrije Universiteit Brussel, VUB, Brussels, Belgium.

Nanobodies are popular and versatile tools for structural biology. They have a compact single immunoglobulin domain organization, bind target proteins with high affinities while reducing their conformational heterogeneity and stabilize multi-protein complexes. Here we demonstrate that engineered nanobodies can also help overcome two major obstacles that limit the resolution of single-particle cryo-electron microscopy reconstructions: particle size and preferential orientation at the water-air interfaces. We have developed and characterized constructs, termed megabodies, by grafting nanobodies onto selected protein scaffolds to increase their molecular weight while retaining the full antigen-binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we demonstrate that megabodies can be used to obtain three-dimensional reconstructions for membrane proteins that suffer from severe preferential orientation or are otherwise too small to allow accurate particle alignment.
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http://dx.doi.org/10.1038/s41592-020-01001-6DOI Listing
January 2021

International Union of Basic and Clinical Pharmacology. CX. Classification of Receptors for 5-hydroxytryptamine; Pharmacology and Function.

Pharmacol Rev 2021 Jan;73(1):310-520

Neuropharmacology Research Group, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom (N.M.B., A.R.); Department of Pharmacology and Therapeutics, School of Biomedical Sciences, Faculty of Medicine, Dentistry and Health Sciences, The University of Melbourne, Parkville, Victoria, Australia (N.M.B., D.H.); Department of Pharmacology, Georgetown University Medical Center, Washington, DC (G.P.A.); Institut de Génomique Functionnelle, Université Montpellier, Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, Montpellier, France (C.B., J.B., S.C.-D., S.C., P.M.); Université de Montpellier, Montpellier, France (C.B., J.B., S.C.-D., S.C., P.M.); C.E.N.T.E.R. Division of Gastroenterology and Hepatology Mayo Clinic, Rochester, Minnesota (M.C.); Center for Addiction Research and Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas (K.A.C., R.M.H.); School of Life Sciences, Medical School, Queen's Medical Centre, The University of Nottingham, Nottingham, United Kingdom (K.C.F.); Department of Pathology and Cell Biology, Columbia University College of Physicians and Surgeons, New York, New York (M.G.); Laboratory of Neurophysiology, Department of Physiology and Biochemistry, Faculty of Medicine and Surgery, University of Malta, Msida, Malta (G.D.G.); Department of Physiology, Department of Obstetrics and Gynaecology, Department of Psychiatry, University of Toronto, Toronto, Ontario, Canada (N.M.G., E.K.L.); Department of Psychiatry, University of California San Diego, La Jolla, California (A.L.H.); Theranyx, Marseille, France (G.H.); Department of Neuroscience and Experimental Therapeutics, Albany Medical College, Albany, New York (K.H.-D.); Ecole Polytechnique Fédérale de Lausanne, Institute of Chemical Sciences and Engineering, Lausanne, Switzerland (R.H., H.V.); Department of Pharmacy-Drug Science, University of Bari Aldo Moro, Bari, Italy (E.L., M.L.); Department of Pharmacology, Faculty of Medicine, University of Oslo and Oslo University Hospital, Oslo, Norway (F.O.L.); Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (S.C.R.L.); INSERM UMR-S 1270, Paris, France (L.M., A.R.); Sorbonne Université, Paris, France (L.M., A.R.); Institut du Fer à Moulin, Paris, France (L.M., A.R.); Drug Development, Grunenthal GmbH, Aachen, Germany (A.C.M.); Tucson, Arizona (D.L.N.); Departments of Psychiatry and Behavioral Sciences and Pharmacology, University of Washington, Seattle, Washington (J.F.N.); Neurolixis Inc., Dana Point, California (A.N.-T.); Université Grenoble Alpes, Institut de Biologie Structurale, Grenoble, France (H.N.); CNRS, Institut de Biologie Structurale, Grenoble, France (H.N.); Commissariat à l'Energie Atomique et aux Energies Alternatives, DSV, Institut de Biologie Structurale, Grenoble, France (H.N.); Department of Pharmacology, University of North Carolina, Chapel Hill, North Carolina (B.L.R.); Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, United Kingdom (G.J.S.); Center for Neuropharmacology and Neuroscience, Albany Medical College, Albany, New York (M.T.); Department of Pharmacology, University of Oxford, Oxford, United Kingdom (T.S.); Cinvestav-Coapa, Pharmacobiology, Mexico City, Tlalpan, Mexico (C.M.V.); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, Michigan (S.W.W.); The Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia (D.H.); and Department of Molecular Medicine, The Scripps Research Institute, La Jolla, California (D.H.).

5-HT receptors expressed throughout the human body are targets for established therapeutics and various drugs in development. Their diversity of structure and function reflects the important role 5-HT receptors play in physiologic and pathophysiological processes. The present review offers a framework for the official receptor nomenclature and a detailed understanding of each of the 14 5-HT receptor subtypes, their roles in the systems of the body, and, where appropriate, the (potential) utility of therapeutics targeting these receptors. SIGNIFICANCE STATEMENT: This review provides a comprehensive account of the classification and function of 5-hydroxytryptamine receptors, including how they are targeted for therapeutic benefit.
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http://dx.doi.org/10.1124/pr.118.015552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770494PMC
January 2021

The Binding of Palonosetron and Other Antiemetic Drugs to the Serotonin 5-HT3 Receptor.

Structure 2020 10 28;28(10):1131-1140.e4. Epub 2020 Jul 28.

CNRS, Univ. Grenoble Alpes, CEA, IBS, 38000 Grenoble, France. Electronic address:

Inaccurately perceived as niche drugs, antiemetics are key elements of cancer treatment alleviating the most dreaded side effect of chemotherapy. Serotonin 5-HT3 receptor antagonists are the most commonly prescribed class of drugs to control chemotherapy-induced nausea and vomiting. These antagonists have been clinically successful drugs since the 1980s, yet our understanding of how they operate at the molecular level has been hampered by the difficulty of obtaining structures of drug-receptor complexes. Here, we report the cryoelectron microscopy structure of the palonosetron-bound 5-HT3 receptor. We investigate the binding of palonosetron, granisetron, dolasetron, ondansetron, and cilansetron using molecular dynamics, covering the whole set of antagonists used in clinical practice. The structural and computational results yield detailed atomic insight into the binding modes of the drugs. In light of our data, we establish a comprehensive framework underlying the inhibition mechanism by the -setron drug family.
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http://dx.doi.org/10.1016/j.str.2020.07.004DOI Listing
October 2020

A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Nat Chem Biol 2019 12 7;15(12):1156-1164. Epub 2019 Oct 7.

Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABA and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.
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http://dx.doi.org/10.1038/s41589-019-0369-4DOI Listing
December 2019

Conformational transitions of the serotonin 5-HT receptor.

Nature 2018 11 31;563(7730):275-279. Epub 2018 Oct 31.

CNRS, Université Grenoble Alpes, CEA, IBS, Grenoble, France.

The serotonin 5-HT receptor is a pentameric ligand-gated ion channel (pLGIC). It belongs to a large family of receptors that function as allosteric signal transducers across the plasma membrane; upon binding of neurotransmitter molecules to extracellular sites, the receptors undergo complex conformational transitions that result in transient opening of a pore permeable to ions. 5-HT receptors are therapeutic targets for emesis and nausea, irritable bowel syndrome and depression. In spite of several reported pLGIC structures, no clear unifying view has emerged on the conformational transitions involved in channel gating. Here we report four cryo-electron microscopy structures of the full-length mouse 5-HT receptor in complex with the anti-emetic drug tropisetron, with serotonin, and with serotonin and a positive allosteric modulator, at resolutions ranging from 3.2 Å to 4.5 Å. The tropisetron-bound structure resembles those obtained with an inhibitory nanobody or without ligand. The other structures include an 'open' state and two ligand-bound states. We present computational insights into the dynamics of the structures, their pore hydration and free-energy profiles, and characterize movements at the gate level and cation accessibility in the pore. Together, these data deepen our understanding of the gating mechanism of pLGICs and capture ligand binding in unprecedented detail.
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http://dx.doi.org/10.1038/s41586-018-0672-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6614044PMC
November 2018

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

Methods Mol Biol 2017 ;1635:139-168

Univ. Grenoble Alpes, CNRS, CEA, CNRS, IBS, F-38000, Grenoble, France.

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.
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http://dx.doi.org/10.1007/978-1-4939-7151-0_8DOI Listing
April 2018

X-ray structure of the mouse serotonin 5-HT3 receptor.

Nature 2014 Aug 3;512(7514):276-81. Epub 2014 Aug 3.

1] Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland [2] Université Grenoble Alpes, IBS, F-38000 Grenoble, France [3] CNRS, IBS, F-38000 Grenoble, France [4] CEA, DSV, IBS, F-38000 Grenoble, France.

Neurotransmitter-gated ion channels of the Cys-loop receptor family mediate fast neurotransmission throughout the nervous system. The molecular processes of neurotransmitter binding, subsequent opening of the ion channel and ion permeation remain poorly understood. Here we present the X-ray structure of a mammalian Cys-loop receptor, the mouse serotonin 5-HT3 receptor, at 3.5 Å resolution. The structure of the proteolysed receptor, made up of two fragments and comprising part of the intracellular domain, was determined in complex with stabilizing nanobodies. The extracellular domain reveals the detailed anatomy of the neurotransmitter binding site capped by a nanobody. The membrane domain delimits an aqueous pore with a 4.6 Å constriction. In the intracellular domain, a bundle of five intracellular helices creates a closed vestibule where lateral portals are obstructed by loops. This 5-HT3 receptor structure, revealing part of the intracellular domain, expands the structural basis for understanding the operating mechanism of mammalian Cys-loop receptors.
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http://dx.doi.org/10.1038/nature13552DOI Listing
August 2014

Sedimentation velocity analytical ultracentrifugation in hydrogenated and deuterated solvents for the characterization of membrane proteins.

Methods Mol Biol 2013 ;1033:219-51

Institut de Biologie Structurale, CEA, Grenoble, France.

This chapter is a step-by-step protocol for setting up, realizing, and analyzing sedimentation velocity experiments in hydrogenated and deuterated solvents, in the context of the characterization of membrane protein, in terms of homogeneity, association state, and amount of bound detergent, based on a real case study of the membrane protein BmrA solubilized in n-Dodecyl-β-D-Maltopyranoside) detergent.
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http://dx.doi.org/10.1007/978-1-62703-487-6_15DOI Listing
March 2014

Large scale expression and purification of the mouse 5-HT3 receptor.

Biochim Biophys Acta 2013 Nov 6;1828(11):2544-52. Epub 2013 Jun 6.

Laboratory of Physical Chemistry of Polymers and Membranes, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.

Receptors of the Cys-loop family are central to neurotransmission and primary therapeutic targets. In order to decipher their gating and modulation mechanisms, structural data is essential. However, structural studies require large amounts of pure, functional receptors. Here, we present the expression and purification of the mouse serotonin 5-HT3 receptor to high purity and homogeneity levels. Inducible expression in human embryonic kidney 293 cells in suspension cultures with orbital shaking resulted in yields of 6-8mg receptor per liter of culture. Affinity purification using a strep tag provided pure protein in active form. Further deglycosylation and removal of the purification tag led to a pentameric receptor after size-exclusion chromatography, at the milligram scale. This material is suitable for crystallography, as demonstrated by X-ray diffraction of receptor crystals at low resolution.
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http://dx.doi.org/10.1016/j.bbamem.2013.05.028DOI Listing
November 2013

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines.

Proc Natl Acad Sci U S A 2012 Oct 3;109(44):E3028-34. Epub 2012 Oct 3.

Department of Cellular and Molecular Medicine, Laboratory of Structural Neurobiology, Catholic University of Leuven, 3000 Leuven, Belgium.

GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.
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http://dx.doi.org/10.1073/pnas.1208208109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3497736PMC
October 2012

A locally closed conformation of a bacterial pentameric proton-gated ion channel.

Nat Struct Mol Biol 2012 May 13;19(6):642-9. Epub 2012 May 13.

Institut Pasteur, Groupe Récepteurs-Canaux, Paris, France.

Pentameric ligand-gated ion channels mediate signal transduction through conformational transitions between closed-pore and open-pore states. To stabilize a closed conformation of GLIC, a bacterial proton-gated homolog from Gloeobacter violaceus whose open structure is known, we separately generated either four cross-links or two single mutations. We found all six mutants to be in the same 'locally closed' conformation using X-ray crystallography, sharing most of the features of the open form but showing a locally closed pore as a result of a concerted bending of all of its M2 helices. The mutants adopt several variant conformations of the M2-M3 loop, and in all cases an interacting lipid that is observed in the open form disappears. A single cross-linked mutant is functional, according to electrophysiology, and the locally closed structure of this mutant indicates that it has an increased flexibility. Further cross-linking, accessibility and molecular dynamics data suggest that the locally closed form is a functionally relevant conformation that occurs during allosteric gating transitions.
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http://dx.doi.org/10.1038/nsmb.2307DOI Listing
May 2012

[X-ray structures of general anesthetics bound to their molecular targets].

Med Sci (Paris) 2011 Dec 23;27(12):1056-7. Epub 2011 Dec 23.

Unité de Dynamique Structurale des Macromolécules, CNRS, URA 2185, Institut Pasteur, 25, rue du Docteur Roux, F-75015 Paris Cedex 15, France.

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http://dx.doi.org/10.1051/medsci/20112712006DOI Listing
December 2011

X-ray structures of general anaesthetics bound to a pentameric ligand-gated ion channel.

Nature 2011 Jan;469(7330):428-31

Institut Pasteur, Groupe Récepteurs-Canaux, F-75015 Paris, France.

General anaesthetics have enjoyed long and widespread use but their molecular mechanism of action remains poorly understood. There is good evidence that their principal targets are pentameric ligand-gated ion channels (pLGICs) such as inhibitory GABA(A) (γ-aminobutyric acid) receptors and excitatory nicotinic acetylcholine receptors, which are respectively potentiated and inhibited by general anaesthetics. The bacterial homologue from Gloeobacter violaceus (GLIC), whose X-ray structure was recently solved, is also sensitive to clinical concentrations of general anaesthetics. Here we describe the crystal structures of the complexes propofol/GLIC and desflurane/GLIC. These reveal a common general-anaesthetic binding site, which pre-exists in the apo-structure in the upper part of the transmembrane domain of each protomer. Both molecules establish van der Waals interactions with the protein; propofol binds at the entrance of the cavity whereas the smaller, more flexible, desflurane binds deeper inside. Mutations of some amino acids lining the binding site profoundly alter the ionic response of GLIC to protons, and affect its general-anaesthetic pharmacology. Molecular dynamics simulations, performed on the wild type (WT) and two GLIC mutants, highlight differences in mobility of propofol in its binding site and help to explain these effects. These data provide a novel structural framework for the design of general anaesthetics and of allosteric modulators of brain pLGICs.
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http://dx.doi.org/10.1038/nature09647DOI Listing
January 2011

Structural approaches of the mitochondrial carrier family.

Methods Mol Biol 2010 ;654:105-17

Institut Pasteur, Unit if Structural Dynamics of Macromolecules, CNRS, URA 2185, Paris, France.

The transport of solutes across the inner mitochondrial membrane is highly selective and necessitates membrane proteins mainly from the mitochondrial carrier family (MCF). These carriers are required for the transport of a variety of metabolites implicated in all the important processes occurring within the mitochondrial matrix. Due to its high abundance, the ADP/ATP carrier (AAC) is the member of the family that was studied most. It is the first mitochondrial carrier for which a high-resolution X-ray structure is known. The carrier was crystallized in the presence of a strong inhibitor, the carboxyatractyloside (CATR). The structure gives an insight not only into the overall fold of mitochondrial carriers in general but also into atomic details of the AAC in a conformation that is open toward the intermembrane space (IMS). Molecular dynamics simulations indicate the first events occurring to the carrier after the binding of ADP. A careful analysis of the primary sequences of all the carriers in light with the structure highlights properties of the protein that are related to the substrate.
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http://dx.doi.org/10.1007/978-1-60761-762-4_6DOI Listing
November 2010

One-microsecond molecular dynamics simulation of channel gating in a nicotinic receptor homologue.

Proc Natl Acad Sci U S A 2010 Apr 22;107(14):6275-80. Epub 2010 Mar 22.

Institut Pasteur, Unit of Structural Dynamics of Macromolecules, Centre National de la Recherche Scientifique Unité de Recherche Associée 2185, Institut Pasteur, Paris, France.

Recently discovered bacterial homologues of eukaryotic pentameric ligand-gated ion channels, such as the Gloeobacter violaceus receptor (GLIC), are increasingly used as structural and functional models of signal transduction in the nervous system. Here we present a one-microsecond-long molecular dynamics simulation of the GLIC channel pH stimulated gating mechanism. The crystal structure of GLIC obtained at acidic pH in an open-channel form is equilibrated in a membrane environment and then instantly set to neutral pH. The simulation shows a channel closure that rapidly takes place at the level of the hydrophobic furrow and a progressively increasing quaternary twist. Two major events are captured during the simulation. They are initiated by local but large fluctuations in the pore, taking place at the top of the M2 helix, followed by a global tertiary relaxation. The two-step transition of the first subunit starts within the first 50 ns of the simulation and is followed at 450 ns by its immediate neighbor in the pentamer, which proceeds with a similar scenario. This observation suggests a possible two-step domino-like tertiary mechanism that takes place between adjacent subunits. In addition, the dynamical properties of GLIC described here offer an interpretation of the paradoxical properties of a permeable A13'F mutant whose crystal structure determined at 3.15 A shows a pore too narrow to conduct ions.
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http://dx.doi.org/10.1073/pnas.1001832107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2852019PMC
April 2010

Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues.

J Physiol 2010 Feb 7;588(Pt 4):565-72. Epub 2009 Dec 7.

Pasteur Institute, G5 Group of Channel-Receptor, CNRS URA 2182, 75015 Paris, France.

Pentameric ligand-gated ion channels (pLGICs) are widely expressed in the animal kingdom and are key players of neurotransmission by acetylcholine (ACh), gamma-amminobutyric acid (GABA), glycine and serotonin. It is now established that this family has a prokaryotic origin, since more than 20 homologues have been discovered in bacteria. In particular, the GLIC homologue displays a ligand-gated ion channel function and is activated by protons. The prokaryotic origin of these membrane proteins facilitated the X-ray structural resolution of the first members of this family. ELIC was solved at 3.3 A in a closed-pore conformation, and GLIC at up to 2.9 A in an apparently open-pore conformation. These data reveal many structural features, notably the architecture of the pore, including its gate and its selectivity filter, and the interactions between the protein and lipids. In addition, comparison of the structures of GLIC and ELIC hints at a mechanism of channel opening, which consists of both a quaternary twist and a tertiary deformation. This mechanism couples opening-closing motions of the channel with a global reorganization of the protein, including the subunit interface that holds the neurotransmitter binding sites in eukaryotic pLGICs.
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http://dx.doi.org/10.1113/jphysiol.2009.183160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2828131PMC
February 2010

Crystal structure of the extracellular domain of a bacterial ligand-gated ion channel.

J Mol Biol 2010 Feb 13;395(5):1114-27. Epub 2009 Nov 13.

Institut Pasteur, Unité de Dynamique Structurale des Macromolécules, CNRS URA 2185, Institut Pasteur, 25 rue du Dr Roux, 75015 Paris, France.

The crystal structure of the extracellular domain (ECD) of the pentameric ligand-gated ion-channel from Gloeobacter violaceus (GLIC) was solved at neutral pH at 2.3 A resolution in two crystal forms, showing a surprising hexameric quaternary structure with a 6-fold axis replacing the expected 5-fold axis. While each subunit retains the usual beta-sandwich immunoglobulin-like fold, small deviations from the whole GLIC structure indicate zones of differential flexibility. The changes in interface between two adjacent subunits in the pentamer and the hexamer can be described in a downward translation by one inter-strand distance and a global rotation of the second subunit, using the first one for superposition. While global characteristics of the interface, such as the buried accessible surface area, do not change very much, most of the atom-atom interactions are rearranged. It thus appears that the transmembrane domain is necessary for the proper oligomeric assembly of GLIC and that there is an intrinsic plasticity or polymorphism in possible subunit-subunit interfaces at the ECD level, the latter behaving as a monomer in solution. Possible functional implications of these novel structural data are discussed in the context of the allosteric transition of this family of proteins. In addition, we propose a novel way to quantify elastic energy stored in the interface between subunits, which indicates a tenser interface for the open form than for the closed form (rest state). The hexameric or pentameric forms of the ECD have a similar negative curvature in their subunit-subunit interface, while acetylcholine binding proteins have a smaller and positive curvature that increases from the apo to the holo form.
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http://dx.doi.org/10.1016/j.jmb.2009.11.024DOI Listing
February 2010

X-ray structure of a pentameric ligand-gated ion channel in an apparently open conformation.

Nature 2009 Jan 5;457(7225):111-4. Epub 2008 Nov 5.

Pasteur Institute, G5 Group of Channel-Receptor, CNRS URA 2182.

Pentameric ligand-gated ion channels from the Cys-loop family mediate fast chemo-electrical transduction, but the mechanisms of ion permeation and gating of these membrane proteins remain elusive. Here we present the X-ray structure at 2.9 A resolution of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel homologue (GLIC) at pH 4.6 in an apparently open conformation. This cationic channel is known to be permanently activated by protons. The structure is arranged as a funnel-shaped transmembrane pore widely open on the outer side and lined by hydrophobic residues. On the inner side, a 5 A constriction matches with rings of hydrophilic residues that are likely to contribute to the ionic selectivity. Structural comparison with ELIC, a bacterial homologue from Erwinia chrysanthemi solved in a presumed closed conformation, shows a wider pore where the narrow hydrophobic constriction found in ELIC is removed. Comparative analysis of GLIC and ELIC reveals, in concert, a rotation of each extracellular beta-sandwich domain as a rigid body, interface rearrangements, and a reorganization of the transmembrane domain, involving a tilt of the M2 and M3 alpha-helices away from the pore axis. These data are consistent with a model of pore opening based on both quaternary twist and tertiary deformation.
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http://dx.doi.org/10.1038/nature07462DOI Listing
January 2009

Mitochondrial bovine ADP/ATP carrier in detergent is predominantly monomeric but also forms multimeric species.

Biochemistry 2008 Nov;47(47):12319-31

CEA, DSV, and CNRS and Universite Joseph Fourier, Institut de Biologie Structurale, 41 rue Jules Horowitz, F-38027, Grenoble, France.

ADP/ATP carriers (AACs) are major and essential constituents of the inner mitochondrial membrane. They drive the import of ADP and the export of newly synthesized ATP. They were described as functional dimers from the 1980s until the structures of the AAC shed doubt on this consensus. We aimed to ascertain the published biophysical data claiming that AACs are dimers and to characterize the oligomeric state of the protein before crystallization. Analytical ultracentrifugation sedimentation velocity experiments clearly show that the bovine AAC is a monomer in 3-laurylamido-N,N'-dimethylpropylaminoxide (LAPAO), whereas in Triton X-100 and reduced Triton X-100, higher molecular mass species can also be identified. Neutron scattering data for monomeric bovine AAC in LAPAO does not give definite conclusions on the association state, because the large amount of detergent and lipids is imperfectly matched by contrast methods. We discuss a possible way to integrate previously published biochemical evidence in favor of assemblies, the lack of well-defined multimers that we observe, and the information from the high-resolution structures, considering supramolecular organizations of AACs within the mitochondrial membrane.
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http://dx.doi.org/10.1021/bi801053mDOI Listing
November 2008

X-ray spectroscopy and X-ray diffraction at wavelengths near the K-absorption edge of phosphorus.

J Synchrotron Radiat 2005 Jul 15;12(Pt 4):402-9. Epub 2005 Jun 15.

Laboratoire d'Enzymologie et de Biochimie Structurales CNRS, Avenue de la Terrasse, 91198 Gif sur Yvette CEDEX, France.

Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.
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http://dx.doi.org/10.1107/S0909049505009222DOI Listing
July 2005