title: Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.
abstract: The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.
authors.affiliation: Department of Biochemistry and Molecular Biology,Victorian Life Sciences Computation Initiative Life Sciences Computation Centre, and.
authors.forename: Itamar
authors.initials: I
authors.lastname: Kass
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: David E
authors.initials: DE
authors.lastname: Hoke
authors.affiliation: Programa de Computação Científica, Fundação Oswaldo Cruz, 21949900 Rio de Janeiro, Brazil;
authors.forename: Mauricio G S
authors.initials: MG
authors.lastname: Costa
authors.affiliation: Department of Biochemistry and Molecular Biology,Australian Research Council Centre of Excellence in Structural and Functional Microbial Genomics, Monash University, Clayton, VIC 3800, Australia;
authors.forename: Cyril F
authors.initials: CF
authors.lastname: Reboul
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Benjamin T
authors.initials: BT
authors.lastname: Porebski
authors.affiliation: Australian Synchrotron, VIC 3168, Australia;
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Cowieson
authors.affiliation: Laboratoire de Biologie et de Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61, F-94235 Cachan, France;
authors.forename: Hervé
authors.initials: H
authors.lastname: Leh
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, 04100 Latina, Italy;
authors.forename: Eugenia
authors.initials: E
authors.lastname: Pennacchietti
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Julia
authors.initials: J
authors.lastname: McCoey
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Oded
authors.initials: O
authors.lastname: Kleifeld
authors.affiliation: Dipartimento di Scienze della Vita e della Riproduzione, Sezione di Chimica Biologica, Facoltà di Medicina e Chirurgia, Università degli Studi di Verona, 37134 Verona, Italy;
authors.forename: Carla
authors.initials: C
authors.lastname: Borri Voltattorni
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst/Sydney, NSW 2010, Australia; and.
authors.forename: David
authors.initials: D
authors.lastname: Langley
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst/Sydney, NSW 2010, Australia; and.
authors.forename: Brendan
authors.initials: B
authors.lastname: Roome
authors.affiliation: Department of Biochemistry and Molecular Biology.
authors.forename: Ian R
authors.initials: IR
authors.lastname: Mackay
authors.affiliation: Garvan Institute of Medical Research, Darlinghurst/Sydney, NSW 2010, Australia; and.
authors.forename: Daniel
authors.initials: D
authors.lastname: Christ
authors.affiliation: Laboratoire de Biologie et de Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61, F-94235 Cachan, France;
authors.forename: David
authors.initials: D
authors.lastname: Perahia
authors.affiliation: Laboratoire de Biologie et de Pharmacologie Appliquée, Ecole Normale Supérieure de Cachan, Centre National de la Recherche Scientifique, 61, F-94235 Cachan, France;
authors.forename: Malcolm
authors.initials: M
authors.lastname: Buckle
authors.affiliation: Dipartimento di Scienze Biochimiche "A. Rossi Fanelli," Sapienza Università di Roma, 00185 Rome, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Paiardini
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Sapienza Università di Roma, 04100 Latina, Italy;
authors.forename: Daniela
authors.initials: D
authors.lastname: De Biase
authors.affiliation: Department of Biochemistry and Molecular Biology, ashley.buckle@monash.edu.
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Buckle
mesh_terms: D001323:Autoantibodies / Q000276:immunology; D015551:Autoimmunity; D003922:Diabetes Mellitus, Type 1 / Q000276:immunology; D005968:Glutamate Decarboxylase / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D006706:Homeostasis / Q000276:immunology*; D006801:Humans; D056004:Molecular Dynamics Simulation; D018377:Neurotransmitter Agents / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D055503:Protein Multimerization; D013329:Structure-Activity Relationship; D005680:gamma-Aminobutyric Acid / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D001323:Autoantibodies; D018377:Neurotransmitter Agents; D005680:gamma-Aminobutyric Acid; D005968:Glutamate Decarboxylase; C401141:glutamate decarboxylase 2
keywords: GABA biosynthesis; autoepitopes; conformational dynamics; immunogenicity; normal mode analysis