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

• Published in: The Journal of biological chemistry Vol. 281; no. 30; pp. 21286 - 21295
• Main Authors: Pecqueur, Ludovic, D'Autréaux, Benoît, Dupuy, Jérome, Nicolet, Yvain, Jacquamet, Lilian, Brutscher, Bernhard, Michaud-Soret, Isabelle, Bersch, Beate
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 28-07-2006; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacterial Proteins - chemistry; Biochemistry; Biochemistry, Molecular Biology; Crystallography, X-Ray - methods; Dimerization; DNA - chemistry; Escherichia coli; Escherichia coli - metabolism; Life Sciences; Magnetic Resonance Spectroscopy - methods; Models, Molecular; Molecular Sequence Data; Protein Denaturation; Protein Folding; Protein Structure, Quaternary; Protein Structure, Secondary; Repressor Proteins - chemistry; Sequence Homology, Amino Acid; Zinc - chemistry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M601278200

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.