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J Biol Chem. 2014 Nov 14;289(46):31765-76. doi: 10.1074/jbc.M114.571208. Epub 2014 Oct 1.

Biochemical and biophysical characterization of the selenium-binding and reducing site in Arabidopsis thaliana homologue to mammals selenium-binding protein 1.

Author information

1
From the Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359.
2
the 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.
3
the 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.
4
the Université Grenoble Alpes, CEA, CNRS, Direction des Sciences du Vivant, Institut de Biologie Structurale, 6 rue Jules Horowitz, F-38044 Grenoble, France.
5
the Université Grenoble Alpes, CNRS & IRD, ISTerre, BP 53, F-38041 Grenoble, France.
6
the 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.
7
the Université Grenoble Alpes, CNRS, Institut NEEL, 25 rue des Martyrs, F-38042 Grenoble, France.
8
From the Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, CEA, Université Grenoble Alpes, CNRS UMR5168, INRA USC1359, Veronique.hugouvieux@cea.fr.

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.

KEYWORDS:

Plant Biochemistry; Protein Chemical Modification; Proteomics; Selenium; X-ray Absorption Spectroscopy

PMID:
25274629
PMCID:
PMC4231655
DOI:
10.1074/jbc.M114.571208
[Indexed for MEDLINE]
Free PMC Article

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