The most studied FUR superfamily member is the ferric uptake regulator Fur, in different microorganisms such as
Escherichia coli,
Helicobacter pylori,
Pseudomonas aeruginosa,
Vibrio cholerae,
Francisella tularensis or
Anabaena sp.. Fur is a transcription factor mainly known as a holo-Fur repressor, that, in the presence of its cofactor Fe2+, controls the expression of almost all genes involved in iron homeostasis (
e.g.,
ent, fec, suf operons involved in biosynthesis of siderophores, iron chelators involved in iron acquisition, their uptake and iron-sulfur biogenesis) but also others still essential
e.g.,
cyoA, fumC, gpmA, nrdH or
sodA which are respectively involved in respiration, tricarboxylic acid cycle, glycolysis, DNA synthesis and resistance to oxidative stress. In fact, Fur is more complex, direct and indirect activating functions as holo- or apo-form activator have been described. For example, Fur is involved in the indirect activation of the expression of the
sodB gene
via the sRNA ryhB (coding for SodB superoxide dismutase) involved in the response to oxidative stress. Direct activation of target as
ftnA and
ftnB genes involved in iron storage may occur. Or its apo-form plays a role in starvation condition to regulate
ymgA,
ymgC and
ariR genes involved in the suppression of biofilm formation and acid resistance. Altogether, many Fur proteins are global regulators that play a central role in bacterial survival.
Moreover, Fur may exist in several stable oligomeric forms, monomer, dimer, and tetramer. Monomeric form can dimerize in the presence of Zn
2+ and reductant. The tetrameric form of Fur is predominant in some species
e.g., PaFur, FtFur and LpFur from
P. aeruginosa,
F. tularensis and
L. pneumophila.
Each subunit of Fur proteins is around 17kDa (145 to 150 amino acids) with two domains. A DNA binding domain (DBD) with a winged helix-turn-helix motif, and a dimerization domain (DD) unfold in monomers but with a long inter subunit β-strand formed in dimers. Most of Fur proteins contain 2 metal binding sites per subunit and a 3
rd accessory site in the DD has been observed in some cases that tunes regulation as in
H. pylori Fur (HpFur). First, the structural site (S1) (Figure 1B), able to bind a zinc ion with 2 to 4 cysteines in same subunit. Then, the highly essential conserved regulatory site (S2) (Figure 1B), involving 3 histidines and 2 glutamic acids, can binds Fe
2+ ion in penta- or hexacoordinate environment (1 Fe atom/subunit in S2 is enough to activate holo-Fur). Other metals can bind in vitro to S2 to activate the protein, such as Ni
2+, Co
2+ or Mn
2+ . Two holo-Fur dimers bind to a palindromic DNA sequence of 19 bp called Fur box, generally present in the -35 and -10 of the promoters of the regulated genes.
Fur's affinity for Fe
2+ is around 10μM, it is about the estimated free iron concentration in a cell. And Fur is present in large amount approximately at 5,000 molecules per cell during exponentially growing cultures. This allows bacteria to respond rapidly to physiological fluctuations in their environment. It has a link with the virulence of pathogenic bacteria, Fur allows to give a signal to bacteria that they are in a host where the environment is limiting in iron and must prepare to attack and defend themselves with virulence factors.
Figure 1. The ferric uptake regulator protein of E. coli.
A. Scheme representation of the action mechanism of EcFur.
B.
E. coli dimeric Fur model with different metal binding sites S1, S2 and S3.
C. Sequence and hypothetical ligands in S1 (underline in green), S2 (underline in yellow) and S3 (underline in cyan), writing on green the 4 cysteines involved in the hypothetic [Fe-S] binding site.
As a global regulator, Fur is therefore an interesting therapeutic target for the research of new antibacterial agents while it does not exist in eukaryote organisms.
This study is about Fur from the commensal and pathogenic bacterium
E. coli (EcFur), it regulates the expression of at least 196 genes. A subunit of EcFur weights 16,795 Da and studies have shown that the protein binds the DNA mainly in its holo-homodimeric form. Full EcFur structure has never been described yet, but only its DBD. An EcFur homology model based on closely related
Vibrio cholerae Fur (VcFur) structure has been built. The Figure 1B represents schematically this model with the three different metal binding-sites. EcFur X-ray absorption studies showed that the zinc ion is coordinated by two cysteines and one or two aspartates or one histidine and one aspartate. The two cysteines, Cys-93 and Cys-96 have been identified as ligands of the zinc atom and are essential to the dimerization. These two cysteines are conserved in a large number of Fur proteins such as EcFur and HpFur. In addition to this CxxC conserved motif, another set of conserved cysteines exist in the C-terminal part of the Fur and Fur-like proteins corresponding either to CxxC such as in HpFur, for example, or to a CX4C motif such as in EcFur (Cys-133 and -138).
Fontenot et al. showed that Fur purified from an
Escherichia coli ΔiscA/ΔsufA mutant strains binds a [2Fe-2S] cluster to 4 cysteines formerly considered as a zinc binding ligand [1-3]. Coupling iron homeostasis and iron-sulfur could make sense and may open new perspectives. We try to decipher the molecular properties of an [2Fe-2S] reconstituted EcFur and to question if iron-sulfur center in EcFur is an artefact or a reality (unpublished results).
[1] Fontenot C.R.
et al. Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in
Escherichia coli.
J. Biol. Chem. 2020, 295, 15454-15463.
[2] Fontenot C.R., Ding H. Biometals. 2022, https://doi.org/10.1007/s10534-022-00390-9
[3] Purcell AG, Fontenot CR, Ding H, Iron-sulfur cluster assembly scaffold protein IscU is required for activation of ferric uptake regulator (Fur) in
Escherichia coli,
Journal of Biological Chemistry. 2024, https://doi.org/10.1016/j.jbc.2024.107142.