Reversible Redox- and Zinc-Dependent Dimerization of the Escherichia coli Fur Protein

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• 作者：Benoî ; t D'Autré ; aux ; Ludovic Pecqueur ; Anne Gonzalez de Peredo ; Rutger E. M. Diederix ; Christelle Caux-Thang ; Lyes Tabet ; Beate Bersch ; Eric Forest ; Isabelle Michaud-Soret
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：February 6, 2007
• 年：2007
• 卷：46
• 期：5
• 页码：1329 - 1342
• 全文大小：396K
• 年卷期：v.46,no.5(February 6, 2007)
• ISSN：1520-4995

文摘

Fur is a bacterial regulator using iron as a cofactor to bind to specific DNA sequences. Thisprotein exists in solution as several oligomeric states, of which the dimer is generally assumed to be thebiologically relevant one. We describe the equilibria that exist between dimeric Escherichia coli Fur andhigher oligomers. The dissociation constant for the dimer-tetramer equilibrium is estimated to be in themillimolar range. Oligomerization is enhanced at low ionic strength and pH. The as-isolated monomericform of Fur is not in equilibrium with the dimer and contains two disulfide bridges (C92-C95 and C132-C137). Binding of the monomer to DNA is metal-dependent and sequence specific with an apparentaffinity 5.5 times lower than that of the dimer. Size exclusion chromatography, EDC cross-linking, andCD spectroscopy show that reconstitution of the dimer from the monomer requires reduction of the disulfidebridges and coordination of Zn²⁺. Reduction of the disulfide bridges or Zn²⁺ alone does not promotedimerization. EDC and DMA cross-links reveal that the N-terminal NH₂ group of one subunit is in anionic interaction with acidic residues of the C-terminal tail and close to Lys76 and Lys97 of the other.Furthermore, the yields of cross-link drastically decrease upon binding of metal in the activation site,suggesting that the N-terminus is involved in the conformational change. Conversely, oxidizing reagents,H₂O₂ or diamide, disrupt the dimeric structure leading to monomer formation. These results establish thatcoordination of the zinc ion and the redox state of the cysteines are essential for holding E. coli Fur in adimeric state.