New insights into the tetrameric family of the Fur metalloregulators

Abstract

The ferric uptake regulator (Fur) belongs to the family of the metal-responsive transcriptional regulators. Fur is a global regulator found in all proteobacteria. It controls the transcription of a wide variety of genes involved in iron metabolism but also in oxidative stress or virulence factor synthesis. As a general view, Fur proteins were considered to be dimeric proteins both in solution and when bound to DNA. However, our recent data demonstrate that Fur proteins can be classified into two subfamilies, according to their quaternary structure. The group of dimers is represented by E. coli, V. cholerae and Y. pestis Fur and the group of highly stable tetramers by P. aeruginosa and F. tularensis Fur. Here, another tetrameric structure of a PaFur mutant containing manganese and zinc metal ions is described. Through biochemical, structural and computational studies, we have deciphered the important structural characteristics of the tetramers and studied the main interactions responsible for their strength. Potential or mean force calculations for tetramer formation have been determinant to quantify these interactions. Moreover calculations allow us to propose that some conserved residues prevent the tetramerization in the subfamily of dimeric Fur.

Ferric uptake regulator (Fur) reversibly binds a [2Fe-2S] cluster to sense intracellular iron homeostasis in Escherichia coli

“…In the past decades, it has been well established that when the intracellular "free" iron content is elevated in bacteria, the global transcription factor ferric uptake regulator (Fur) binds "free" ferrous iron to repress the genes encoding for iron uptake systems and to stimulate the genes encoding for iron storage proteins in bacteria (5)(6)(7)(8)(9). Although the purified E. coli Fur has been reconstituted with ferrous iron in vitro (41,43), the "iron-bound" Fur has never been isolated from E. coli or any other bacteria.…”

“…Thus, it is most likely that Fur binds a [2Fe-2S] cluster (not a [4Fe-4S] cluster or a mononuclear iron) when the intracellular "free" iron content is elevated in the E. coli iscA/sufA mutant cells. Use of a [2Fe-2S] cluster in Fur to sense the intracellular "free" iron content may represent physiological connections between intracellular iron homeostasis and regulation of acid tolerance, oxidative stress response and bacterial virulence (8), since assembly of the [2Fe-2S] cluster in Fur requires not only the intracellular "free" iron but also sulfide that is derived from L-cysteine by cysteine desulfurase IscS (17). In this context, we propose that while elevation of the intracellular "free" iron content together with available sulfide leads to assembly of a [2Fe-2S] cluster in Fur and formation of an active Fur repressor in cells, depletion of the intracellular "free" iron content results in disassembly of the [2Fe-2S] cluster in Fur and inactivates Fur as a repressor.…”

“…The bacterial intracellular "free" iron concentration is primarily regulated by a global transcription factor Ferric uptake regulator (Fur) (1)(2)(3)(4). It has been generally assumed that when the intracellular "free" iron concentration is elevated, Fur binds "free" ferrous iron and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins (5)(6)(7)(8)(9). The crystallographic studies of the Fur proteins from Escherichia coli (10), Mycobacterium tuberculosis (11), Vibrio cholerae (12), Helicobacter pylori (13), Campylobacter jejuni (14), and Francisella tularensis (15) have revealed that Fur protein exists as a homodimer or tetramer (8) with each monomer containing three putative metal binding sites.…”

“…It has been generally assumed that when the intracellular "free" iron concentration is elevated, Fur binds "free" ferrous iron and the iron-bound Fur represses the genes encoding for iron uptake systems and stimulates the genes encoding for iron storage proteins (5)(6)(7)(8)(9). The crystallographic studies of the Fur proteins from Escherichia coli (10), Mycobacterium tuberculosis (11), Vibrio cholerae (12), Helicobacter pylori (13), Campylobacter jejuni (14), and Francisella tularensis (15) have revealed that Fur protein exists as a homodimer or tetramer (8) with each monomer containing three putative metal binding sites. The first metal binding site (site 1) is coordinated by His-87, Asp-89, Glu-108, and His-125 (the residue numbers are based on the E. coli Fur), while the second site (site 2) is coordinated by His-33, Glu-81, His-88, and His-90 (12).…”

Biochemical characterization of ferric uptake regulator (Fur) from Aliivibrio salmonicida. Mapping the DNA sequence specificity through binding studies and structural modelling

“…The sequence alignment between these homologs and AsFur highlights several conserved sequence patches both within the DNA-binding- and dimerization domains (DBD and DD, respectively). Although most Fur proteins characterised are found to be dimers in solution, some also exist in the form of stable tetramers, exemplified by Fur from Francisella tularensis and Pseudomonas aeruginosa (Nader et al 2019 ; Perard et al 2018 ). While the conserved positions in the DD are mainly attributed to metal-coordination, the conserved patches in the DBD are involved in interactions with the Fur box (Fig.…”