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 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.

Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis.

Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue.

Spectroscopic and saturation magnetization properties of the manganese- and cobalt-substituted Fur (ferric uptake regulation) protein from Escherichia coli.

The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.

Synthesis and biological evaluation of flavanones and flavones related to podophyllotoxin.

A series of novel flavonoids comprising structural elements present in the antineoplastic agents podophyllotoxin and etoposide was synthesized. These oxygen-containing analogues of antiproliferative quinolones moderate cytotoxicity towards L1210 and HT-29 cell lines.