Conformational Changes of the Ferric Uptake Regulation Protein Upon Metal Activation and DNA Binding; First Evidence of Structural Homologies With the Diphtheria Toxin Repressor

J Mol Biol. 2001 Jun 29;310(1):83-91. doi: 10.1006/jmbi.2001.4769.

Abstract

Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain.

MeSH terms

  • Amino Acid Sequence
  • Apoproteins / chemistry
  • Apoproteins / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / metabolism*
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism
  • Deuterium / metabolism
  • Helix-Turn-Helix Motifs
  • Imidoesters / metabolism
  • Iron / metabolism*
  • Lysine / chemistry
  • Lysine / metabolism
  • Manganese / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Binding
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Repressor Proteins / chemistry*
  • Repressor Proteins / metabolism*
  • Sequence Alignment
  • Spectrometry, Mass, Electrospray Ionization

Substances

  • Apoproteins
  • Bacterial Proteins
  • DNA-Binding Proteins
  • DtxR protein, Corynebacterium diphtheriae
  • Imidoesters
  • Repressor Proteins
  • ferric uptake regulating proteins, bacterial
  • Manganese
  • DNA
  • Deuterium
  • methyl acetimidate
  • Iron
  • Lysine