The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites

Authors

  • Cyril Dian,

    1. Structural Biology Group, European Synchrotron Radiation Facility, BP 220 F-38043 Grenoble cedex 9, France.
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    • Present addresses: Laboratoire de Mécanismes Moléculaires des Infections et Pathologies, Institut de Biologie Structurale (IBS), CNRS UMR 5075, Université Joseph Fourier, CEA DSV, 41 rue Jules Horowitz, Grenoble F-38027, France;

    • These authors contributed equally to this work.

  • Sylvia Vitale,

    1. CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux; Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV); Université Joseph Fourier, 17 avenue des Martyrs, Grenoble F-38054 cedex 9, France.
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    • Gene Center and Department of Biochemistry, Ludwig Maximilians University, Feodor-Lynen-Strasse 25, D-81377 Munich, Germany;

    • These authors contributed equally to this work.

  • Gordon A. Leonard,

    1. Structural Biology Group, European Synchrotron Radiation Facility, BP 220 F-38043 Grenoble cedex 9, France.
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  • Christelle Bahlawane,

    1. CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux; Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV); Université Joseph Fourier, 17 avenue des Martyrs, Grenoble F-38054 cedex 9, France.
    2. Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany.
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  • Caroline Fauquant,

    1. CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux; Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV); Université Joseph Fourier, 17 avenue des Martyrs, Grenoble F-38054 cedex 9, France.
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    • Laboratoire d'Enzymologie et Biochimie Structurales UPR3082 CNRS, Gif-sur-Yvette, France. Bât 34,Rue de la terrasse, 91198 Gif sur Yvette, France.

  • Damien Leduc,

    1. Institut Pasteur, Unité Pathogenèse de Helicobacter, 28 rue Docteur Roux, 75724 Paris Cedex 15, France.
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  • Cécile Muller,

    1. Institut Pasteur, Unité Pathogenèse de Helicobacter, 28 rue Docteur Roux, 75724 Paris Cedex 15, France.
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  • Hilde de Reuse,

    1. Institut Pasteur, Unité Pathogenèse de Helicobacter, 28 rue Docteur Roux, 75724 Paris Cedex 15, France.
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  • Isabelle Michaud-Soret,

    Corresponding author
    1. CNRS UMR 5249 Laboratoire de Chimie et Biologie des Métaux; Commissariat à l'Energie Atomique (CEA), Direction des Sciences du Vivant (DSV), Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV); Université Joseph Fourier, 17 avenue des Martyrs, Grenoble F-38054 cedex 9, France.
      E-mail laurent.terradot@ibcp.fr; Tel. (+33) 4 72 72 26 52; Fax (+33) 4 72 72 26 04 and E-mail imichaud@cea.fr; Tel (+33) 4 38 78 99 40; Fax (+33) 4 38 78 54 87.
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  • Laurent Terradot

    Corresponding author
    1. Structural Biology Group, European Synchrotron Radiation Facility, BP 220 F-38043 Grenoble cedex 9, France.
    2. Institut de Biologie et Chimie des Protéines, Biologie Structurale des Complexes Macromoléculaires Bactériens, UMR 5086 CNRS Université de Lyon, IFR128, 7 Passage du Vercors, F-69367 Lyon Cedex 07, France
      E-mail laurent.terradot@ibcp.fr; Tel. (+33) 4 72 72 26 52; Fax (+33) 4 72 72 26 04 and E-mail imichaud@cea.fr; Tel (+33) 4 38 78 99 40; Fax (+33) 4 38 78 54 87.
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E-mail laurent.terradot@ibcp.fr; Tel. (+33) 4 72 72 26 52; Fax (+33) 4 72 72 26 04 and E-mail imichaud@cea.fr; Tel (+33) 4 38 78 99 40; Fax (+33) 4 38 78 54 87.

Summary

Fur, the ferric uptake regulator, is a transcription factor that controls iron metabolism in bacteria. Binding of ferrous iron to Fur triggers a conformational change that activates the protein for binding to specific DNA sequences named Fur boxes. In Helicobacter pylori, HpFur is involved in acid response and is important for gastric colonization in model animals. Here we present the crystal structure of a functionally active HpFur mutant (HpFur2M; C78S-C150S) bound to zinc. Although its fold is similar to that of other Fur and Fur-like proteins, the crystal structure of HpFur reveals a unique structured N-terminal extension and an unusual C-terminal helix. The structure also shows three metal binding sites: S1 the structural ZnS4 site previously characterized biochemically in HpFur and the two zinc sites identified in other Fur proteins. Site-directed mutagenesis and spectroscopy analyses of purified wild-type HpFur and various mutants show that the two metal binding sites common to other Fur proteins can be also metallated by cobalt. DNA protection and circular dichroism experiments demonstrate that, while these two sites influence the affinity of HpFur for DNA, only one is absolutely required for DNA binding and could be responsible for the conformational changes of Fur upon metal binding while the other is a secondary site.

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