title: Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.
abstract: The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3(2-)) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3(2-) to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
authors.forename: Florie
authors.initials: F
authors.lastname: Schild
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Biologie à Grande Echelle, Université Grenoble Alpes, CEA, INSERM, 17 rue des Martyrs, F-38000 Grenoble, France.
authors.forename: Sylvie
authors.initials: S
authors.lastname: Kieffer-Jaquinod
authors.affiliation: European Molecular Biology Laboratory Outstation, 71 avenue des Martyrs, F-38042 Grenoble, France and Unit for Virus Host-Cell Interactions, Université Grenoble Alpes-EMBL-CNRS, 71 avenue des Martyrs, 38042 France.
authors.forename: Andrés
authors.initials: A
authors.lastname: Palencia
authors.affiliation: Université Grenoble Alpes, CEA, CNRS, Direction des Sciences du Vivant, Institut de Biologie Structurale, 6 rue Jules Horowitz, F-38044 Grenoble, France.
authors.forename: David
authors.initials: D
authors.lastname: Cobessi
authors.affiliation: Université Grenoble Alpes, CNRS & IRD, ISTerre, BP 53, F-38041 Grenoble, France.
authors.forename: Géraldine
authors.initials: G
authors.lastname: Sarret
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
authors.forename: Chloé
authors.initials: C
authors.lastname: Zubieta
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
authors.forename: Agnès
authors.initials: A
authors.lastname: Jourdain
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
authors.forename: Renaud
authors.initials: R
authors.lastname: Dumas
authors.affiliation: Laboratoire de Chimie et Biologie des Métaux, Université Grenoble Alpes, CEA, CNRS, Institut de Recherches en Technologies et Sciences pour le Vivant, 17 rue des Martyrs, F-38000 Grenoble, France, and.
authors.forename: Vincent
authors.initials: V
authors.lastname: Forge
authors.affiliation: Université Grenoble Alpes, CNRS, Institut NEEL, 25 rue des Martyrs, F-38042 Grenoble, France.
authors.forename: Denis
authors.initials: D
authors.lastname: Testemale
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
authors.forename: Jacques
authors.initials: J
authors.lastname: Bourguignon
authors.affiliation: Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359,. Electronic address: Veronique.hugouvieux@cea.fr.
authors.forename: Véronique
authors.initials: V
authors.lastname: Hugouvieux
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000187:drug effects*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D018506:Gene Expression Regulation, Plant; D006801:Humans; D008024:Ligands; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012643:Selenium / Q000737:chemistry*; D051137:Selenium-Binding Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D008024:Ligands; D011994:Recombinant Proteins; C545453:SBP1 protein, Arabidopsis; C495175:SELENBP1 protein, human; D051137:Selenium-Binding Proteins; D012643:Selenium; D003545:Cysteine
keywords: Plant Biochemistry; Protein Chemical Modification; Proteomics; Selenium; X-ray Absorption Spectroscopy