Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor

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 sug gested that metal binding induces a conformational change in the protein, w hich is subsequently able to recognize DNA. This mechanism of activation ha s been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protei n was studied, first in the apo form of the protein, then after metal activ ation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of targ et DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On t he 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 confor mational modification of this specific motif. The recognition helix, intera cting 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. Th ese results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentrati on regulators, and the structure of their DNA-binding domain. (C) 2001 Acad emic Press.