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J. Biol. Chem., Vol. 281, Issue 30, 21286-21295, July 28, 2006

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Structural Changes of Escherichia coli Ferric Uptake Regulator during Metal-dependent Dimerization and Activation Explored by NMR and X-ray Crystallography^*

Ludovic Pecqueur, Benoît D'Autréaux, Jérome Dupuy^¶, Yvain Nicolet^¶, Lilian Jacquamet^¶, Bernhard Brutscher, Isabelle Michaud-Soret¹, and Beate Bersch²

From the Laboratoire de Résonance Magnétique Nucléaire des Protéines and ^¶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 and 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

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 1.8Å 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.

Received for publication, February 9, 2006 , and in revised form, April 21, 2006.

The atomic coordinates and structure factors (code 2FU4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Fig. S1.

¹ To whom correspondence may be addressed: Laboratoire de Physicochimie des Métaux en Biologie, Département de Réponse et Dynamique Cellulaires, CEA-Grenoble, 17 Ave. des Martyrs, 38054 Grenoble Cedex 9, France. Tel.: 33-4-38-78-99-40; Fax: 33-4-38-78-34-62; E-mail: imichaud{at}cea.fr.

² To whom correspondence may be addressed: Laboratoire de Résonance Magnétique Nucléaire, Institut de Biologie Structurale, 41 Rue Jules Horowitz, 38027 Grenoble Cedex 1, France. Tel.: 33-4-38-78-48-25; Fax: 33-4-38-78-54-94; E-mail: beate.bersch{at}ibs.fr.

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