title: Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography.
abstract: Ferric uptake regulator (Fur) is a global bacterial regulator that uses iron as a cofactor to bind to specific DNA sequences. Escherichia coli Fur is usually isolated as a homodimer with two metal sites per subunit. Metal binding to the iron site induces protein activation; however the exact role of the structural zinc site is still unknown. Structural studies of three different forms of the Escherichia coli Fur protein (nonactivated dimer, monomer, and truncated Fur-(1-82)) were performed. Dimerization of the oxidized monomer was followed by NMR in the presence of a reductant (dithiothreitol) and Zn(II). Reduction of the disulfide bridges causes only local structure variations, whereas zinc addition to reduced Fur induces protein dimerization. This demonstrates for the first time the essential role of zinc in the stabilization of the quaternary structure. The secondary structures of the mono- and dimeric forms are almost conserved in the N-terminal DNA-binding domain, except for the first helix, which is not present in the nonactivated dimer. In contrast, the C-terminal dimerization domain is well structured in the dimer but appears flexible in the monomer. This is also confirmed by heteronuclear Overhauser effect data. The crystal structure at 1.8A resolution of a truncated protein (Fur-(1-82)) is described and found to be identical to the N-terminal domain in the monomeric and in the metal-activated state. Altogether, these data allow us to propose an activation mechanism for E. coli Fur involving the folding/unfolding of the N-terminal helix.
authors.affiliation: Laboratoire de Physicochimie des Métaux en Biologie (Unité Mixte de Recherche 5155 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), Département Réponse et Dynamique Cellulaires, Commissariat à l'Energie Atomique-Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France; Laboratoire de Résonance Magnétique Nucléaire des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1.
authors.forename: Ludovic
authors.initials: L
authors.lastname: Pecqueur
authors.affiliation: Laboratoire de Physicochimie des Métaux en Biologie (Unité Mixte de Recherche 5155 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), Département Réponse et Dynamique Cellulaires, Commissariat à l'Energie Atomique-Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France.
authors.forename: Benoît
authors.initials: B
authors.lastname: D'Autréaux
authors.affiliation: Laboratoire de Cristallographie et de Cristallogénèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1.
authors.forename: Jérome
authors.initials: J
authors.lastname: Dupuy
authors.affiliation: Laboratoire de Cristallographie et de Cristallogénèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1.
authors.forename: Yvain
authors.initials: Y
authors.lastname: Nicolet
authors.affiliation: Laboratoire de Cristallographie et de Cristallogénèse des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1.
authors.forename: Lilian
authors.initials: L
authors.lastname: Jacquamet
authors.affiliation: Laboratoire de Résonance Magnétique Nucléaire des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Brutscher
authors.affiliation: Laboratoire de Physicochimie des Métaux en Biologie (Unité Mixte de Recherche 5155 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), Département Réponse et Dynamique Cellulaires, Commissariat à l'Energie Atomique-Grenoble, 17 Avenue des Martyrs, F-38054 Grenoble Cedex 9, France. Electronic address: imichaud@cea.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Michaud-Soret
authors.affiliation: Laboratoire de Résonance Magnétique Nucléaire des Protéines, Institut de Biologie Structurale Jean-Pierre Ebel (Unité Mixte de Recherche 5075 CNRS/Commissariat à l'Energie Atomique/Université Joseph Fourier), F-38027 Grenoble Cedex 1. Electronic address: beate.bersch@ibs.fr.
authors.forename: Beate
authors.initials: B
authors.lastname: Bersch
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D018360:Crystallography, X-Ray / Q000379:methods; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D015032:Zinc / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D004247:DNA; D015032:Zinc