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Review

Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur)

by
Sung-Min Kang
1,†,
Hoon-Seok Kang
2,†,
Woo-Hyun Chung
1,
Kyu-Tae Kang
1 and
Do-Hee Kim
3,*
1
College of Pharmacy, Duksung Women’s University, Seoul 01369, Republic of Korea
2
Interdisciplinary Graduate Program in Advanced Convergence Technology & Science, Jeju National University, Jeju 63243, Republic of Korea
3
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Sookmyung Women’s University, Seoul 04310, Republic of Korea
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Biomolecules 2024, 14(8), 981; https://doi.org/10.3390/biom14080981 (registering DOI)
Submission received: 27 June 2024 / Revised: 27 July 2024 / Accepted: 3 August 2024 / Published: 9 August 2024
(This article belongs to the Topic Metalloproteins and Metalloenzymes)
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Abstract

:
Iron is crucial for the metabolism and growth of most prokaryotic cells. The ferric uptake regulator (Fur) protein plays a central role in regulating iron homeostasis and metabolic processes in bacteria. It ensures the proper utilization of iron and the maintenance of cellular functions in response to environmental cues. Fur proteins are composed of an N-terminal DNA-binding domain (DBD) and a C-terminal dimerization domain (DD), typically existing as dimers in solution. Fur proteins have conserved metal-binding sites named S1, S2, and S3. Among them, site S2 serves as a regulatory site, and metal binding at S2 results in conformational changes. Additionally, as a transcriptional regulator, Fur specifically binds to a consensus DNA sequence called the Fur box. To elucidate the structural and functional properties of Fur proteins, various structures of metal- or DNA-bound Fur proteins or apo-Fur proteins have been determined. In this review, we focus on the structural properties of Fur proteins according to their ligand-bound state and the drug development strategies targeting Fur proteins. This information provides valuable insights for drug discovery.

1. Introduction

Iron, the fourth most abundant element in the solid outer crust, is crucial for the metabolism and growth of most prokaryotic cells, except for Lactobacillus and Borrelia burgdorferi [1,2,3,4]. Microorganisms tend to have increased iron availability under anaerobic conditions but decreased availability under aerobic conditions, as iron exists in the form of Fe3+, which is easily oxidized and converted to insoluble hydroxides [5].
Iron availability is crucial because it affects various cellular processes, including nitrogen fixation, energy metabolism, DNA synthesis, and oxygen transport, by providing an enzymatic cofactor and participating in electron transfer reactions [6,7,8]. Iron deficiency can cause stress and abnormal changes in cell metabolism and growth, while iron excess can lead to toxicity due to the generation of reactive oxygen species via the Fenton reaction and the production of hydroxyl free radicals through the Haber-Weiss process in cellular metabolism [9]. Therefore, microorganisms have evolved complex mechanisms to regulate iron levels to meet metabolic needs while minimizing the risk of toxicity. Among these mechanisms, the ferric uptake regulator (Fur) is known to be the most important regulator of iron levels [10,11].
Fur is a regulator found in most prokaryotes that mainly regulates iron response genes and is involved in various aspects of prokaryotic metabolism [12]. The term “Fur” was first used to name the outer membrane proteins FhuA, FecA, Cir, 76K, and 83K produced in mutant strains by studying the genes related to the iron uptake system according to iron concentration in anaerobic conditions in Escherichia coli [13]. Additional studies have indicated that several divalent metal ions can bind to the Fur protein similarly to iron, thereby regulating the operon. This confirms the existence of several metalloregulators in addition to Fur [14]. Therefore, the group of metalloregulators is referred to as the Fur family, which can be classified into three main types based on their primary response: metal availability, peroxide stress, and heme availability [15]. This review will focus on the metal-binding and structural properties, functions, and applications in drug discovery of Fur proteins among Fur family proteins.
This review aims not only to introduce the regulatory mechanism of Fur but also to describe the structural conformational changes induced by metal binding. This analysis is crucial for future targeted drug discovery involving the Fur protein and provides a new perspective absent from previous reviews. While other review articles are available, they typically focus on reporting phenomena and introducing related proteins. Even when structural analysis is included, it is limited to fragmented descriptions of static states. In contrast, this review emphasizes the structural differences based on ligand-binding states. This specific focus on the structural properties of Fur proteins offers valuable insights for drug discovery targeting the Fur protein.

2. The Roles of Fur

The classic role of Fur is generally known as a repressor. Apo Fur exists as a dimer in solution. When iron is abundant, the Fur protein binds to Fe2+, causing a conformational change. When a metal binds to the regulatory site, it causes the DBD to rotate relative to the DD, increasing the space between the two DBDs, thereby allowing DNA to be accommodated without steric hindrance [16,17]. The preformed dimers inhibit transcription by binding to target promoters called Fur binding sites (Fur boxes) in their dimer state. Conversely, when iron is deficient, Fur releases the bound Fe2+ and dissociates from the target promoter, allowing the site to initiate transcription by binding RNA polymerase [18].
In addition to the classical role of Fe-Fur repression described above, various models describe Fur acting as Fe-Fur activation, apo-Fur repression, and apo-Fur activation [19] (Figure 1). Fur functions as a repressor or activator depending on whether it is bound to Fe under iron-rich conditions (Fe-Fur) or not bound to Fe under iron-deficient conditions (apo-Fur). The difference between repression and activation lies in that repression prevents the binding of RNA polymerase to Fur boxes near the transcription start site, while activation facilitates binding [20].
In summary, the role of Fur as a repressor or activator is closely linked to the position of the Fur box relative to the RNA polymerase binding site. Additionally, Fur’s regulatory functions are further modulated by interactions with sRNAs, nucleoid-associated proteins, co-repressors, or co-activators, as well as other regulatory mechanisms such as allosteric regulation and feedback loops [18,19,20].
Each of the four models regulates different genes. For example, Helicobacter pylori Fur utilizes all four models mentioned earlier [21]. Firstly, Fe-Fur represses the transcription of iron uptake genes such as frpB by binding to promoters when iron is abundant [22]. Secondly, Fe-Fur has been found to activate the expression of oorDABC [23], nifS [24], and cagA [25] under iron-rich conditions. Thirdly, apo-Fur inhibits the synthesis of prokaryotic ferritin (Pfr), an iron storage protein, when iron concentration is low [26], and superoxide dismutase (SodB), which converts toxic superoxide radicals into oxygen and hydrogen peroxide, is expressed in iron-rich conditions but not in iron-limited conditions due to apo-Fur repression [27]. Fourthly, when iron concentration is low, apo-Fur activation causes mutations in the iron-binding site of Fur, leading to the activation of Fur transcription by binding to the promoter (Pfur) as it oligomerizes regardless of iron presence [28].
In addition to its classic role as a transcription regulator, Fur exhibits significant variability in its regulatory functions across different bacterial species. For instance, in Salmonella, Fur regulates the Salmonella pathogenicity island (SPI)-1, facilitating bacterial invasion, highlighting its role in virulence regulation [29]. In Edwardsiella piscicida, Fur controls siderophore production and expression of other critical molecules, demonstrating its involvement in iron acquisition and metabolic regulation [30]. Furthermore, in E. coli, Fur influences the expression of 81 genes across 42 transcription units, linking iron homeostasis with DNA synthesis, energy metabolism, and biofilm development, thus showcasing its extensive regulatory network and adaptive responses to iron availability [31]. These diverse roles underscore the importance of Fur in coordinating cellular processes related to iron metabolism and stress responses, providing valuable insights for drug discovery targeting Fur proteins across various bacterial pathogens.

3. Structural Characteristics of Fur

An analysis of PDB depositions as of 2024 reveals approximately 40 Fur proteins and paralogs, including Mur, Nur, PerR, and Zur. Among these, thirteen Fur protein-related structures from bacteria such as Campylobacter jejuni [32,33], E. coli [34], Francisella tularensis [35], H. pylori [5], Magnetospirillum gryphiswaldense [36], Pseudomonas aeruginosa [37], and Vibrio cholerae [38] have been identified through crystallographic analysis. These structures reveal Fur proteins as dimers or tetramers, each with three putative metal-binding sites per monomer (Table 1). Most structures are metal-bound, some are metal-free, and others are DNA-bound [12].
Fur proteins are typically composed of two distinct domains: an N-terminal DBD and a C-terminal DD, linked by a hinge loop [39,40] (Figure 2). All determined three-dimensional structures of Fur proteins contain these two domains. The DBD includes a helix-turn-helix motif and consists of four consecutive α-helices followed by two β-strands (α1-α2-α3-α4-β1-β2), while the DD comprises a mixed α/β domain with α5 situated between β4 and β5 (β3-β4-α5-β5-α6). Dimerization of Fur occurs primarily through an antiparallel β-sheet involving α5 and β5, resulting in a V-shaped dimer.
Since the initial observation of P. aeruginosa Fur, most three-dimensional structures of Fur proteins have been found in metal-bound forms. The first metal-free structure was identified in M. gryphiswaldense Fur (PDB ID: 4RAY) [36]. Notably, the metal-binding sites of purified and structurally determined Fur proteins are typically occupied by Zn2+ or other metal ions, despite Fur being a ferric uptake regulator responsive to iron. “Iron-bound” Fur proteins have rarely been isolated from most bacteria. It is important to note that there has been no direct in vivo evidence of iron binding in Fur proteins in any bacteria. For instance, the iron-bound structure of F. tularensis Fur (PDB ID: 5NHK) has only been observed through in vitro reconstitution with iron, not in vivo [35]. The lack of direct in vivo evidence of iron binding in Fur proteins might be due to the transient nature of metal binding and the dynamic changes in intracellular metal concentrations. Given that the intracellular iron level is regulated by Fur, it is challenging to elevate the intracellular free iron concentration in bacteria without deleting Fur [41]. Additionally, the metal-binding study of E. coli Fur reported the dissociation constants ranging from 0.14 nM to 24 μM, with the order of Zn2+ > Co2+ > Fe2+ > Mn2+ [42]. Another study on the binding of E. coli Fur to Mn2+ or Fe2+ revealed the dissociation constants of 1.15 μM and 1.25 μM, respectively [43]. The relatively weak binding affinity of E. coli Fur to Fe2+ might also contribute to the absence of the iron-bound Fur in vivo [44,45].

4. Metal Binding Sites of Fur and Its Roles

The initial Fur structure identified was a two-metal-bound structure from P. aeruginosa [37], and most subsequent structures were also two-metal-bound forms. The first structure identified with three metals bound was from H. pylori Fur [5]. In addition to H. pylori, other paralogs of Fur family proteins, which exhibit three metal-binding sites, include Streptomyces coelicolor Zur [46] and Mycobacterium tuberculosis Zur (FurB) [47]. These metal-binding sites are labeled S1, S2, and S3, and the sites are highly conserved in sequence, as shown in Figure 3.
Site S1 is coordinated by four sulfur atoms from two pairs of cysteines in CX2C motifs. It connects the short C-terminal helix α5 to the β-sheet (β3-β4-β5) of the dimerization domain (Figure 2 and Figure 3). Site S1 is known to be of significant importance for maintaining the structural integrity of the protein and dimerization in Fur proteins. In E. coli, reconstitution of the dimer from the Fur monomer requires the reduction of the Cys92-Cys95 disulfide bridges and coordination of Zn2+ [50]. Among the Fur structures, only those of C. jejuni, F. tularensis, and H. pylori Fur proteins have Zn2+ bound to site S1. It can be concluded that no metal is present in site S1 of M. gryphiswaldense and P. aeruginosa Fur proteins, due to the absence of the two Cys residues in the C-terminal CX2C motif.
Site S2 is a nitrogen/oxygen-rich site with ligands such as His on the loop between α2 and α3 and Glu on β2, two His on β3, and Glu on β4. Site S2 connects the DBD, which provides two ligands (His and Glu) (Figure 2 and Figure 3), and the DD, which provides two to three ligands (two His and Glu). The following bacteria have been shown to possess the site S2-bound metal: F. tularensis, H. pylori, M. gryphiswaldense, P. aeruginosa, and V. cholerae (Table 1). In contrast to site S1, which demonstrated the ability to bind solely Zn2+, site S2 exhibited the property to bind Fe2+ or Mn2+ besides Zn2+, depending on the organisms. The conserved residues His and Glu, which are involved as ligands for the metal ion, exhibit different coordination properties depending on the bacterial species. This location is crucial for maintaining the overall structure of the protein. In the case of P. aeruginosa Fur, the absence of zinc binding to this site may result in the partial unfolding or aggregation of the protein, which could consequently impair its function [37].
The significance of the observed variations in geometry at site S2 is crucial for regulating the function of Fur proteins across diverse bacteria. For instance, while in some bacteria site S2 binds exclusively to zinc; in others, it can also bind to different metals, such as iron or manganese. This allows Fur proteins to adapt to varying metal ion concentrations in the bacterial environment, thereby regulating cellular iron levels. The reason for this variation in metal binding is believed to be related to the specific structural environment in which each metal ion exists most securely. Thus, the interaction of Fur proteins with specific metal ions can vary depending on how these ions bind to the S2 site [5,37].
Site S2 is the iron-sensing site and serves as a regulatory site, responsible for triggering the conformational change required to activate the Fur protein, enabling it to bind specifically to DNA sequences [5,11]. The overlay of the identified Fur structures reveals a high degree of structural overlap. However, the structures lacking metal in the site S2 do not overlap with the structure mentioned above.
The structures of Fur proteins from C. jejuni, H. pylori, M. gryphiswaldense, P. aeruginosa, and V. cholerae exhibited a metal-binding site S3. Site S3 is located towards the DD of Fur proteins, between the β3, β4, and β5 strands and helix α5 (Figure 2 and Figure 3). The residues His and Asp on β3, Glu on α5, and His on β5 are typically involved in metal ion coordination. In this site, it was found that Zn2+ was bound to the Fur proteins, with the exception of M. gryphiswaldense Fur, which was found to be bound to Mn2+. The coordination of metal ions at site S3 also varies, with the coordination geometry being either tetrahedral (H. pylori and V. cholerae Fur) or octahedral (C. jejuni, M. gryphiswaldense, and P. aeruginosa Fur). Although metal bound in site S3 is not essential for DNA binding, metal binding at this site significantly enhances the affinity for DNA [5].
Recent studies have demonstrated that Fur proteins can bind to [2Fe-2S] clusters via the conserved Cys93 and Cys96 (corresponding to Cys102 and Cys105 in the case of H. pylori Fur) when the intracellular free iron content is elevated. The following order of binding affinity for the [2Fe-2S] clusters was observed: H. influenzae Fur, E. coli Fur, V. cholerae Fur, and H. pylori Fur. M. gryphiswaldense Fur, which lacks residues Cys93 and Cys96, is unable to form a complex with [2Fe-2S] clusters [51]. The binding of Fur to the [2Fe-2S] cluster was found to be unstable, with iron being rapidly released upon reduction. This indicates that Fur binds reversibly to the [2Fe-2S] cluster, contingent on the concentration of free iron within the bacterial cell [51]. Further experiments utilizing E. coli and H. influenzae Fur constructs have demonstrated that the C-terminal domain of Fur binds a [2Fe-2S] cluster [52]. Typically, [2Fe-2S] clusters are involved in the active form of the Fur protein, and their binding is regulated by intracellular iron levels. Therefore, the association with [2Fe-2S] clusters is expected to play a crucial role in Fur’s function and intracellular iron level regulation. Moreover, the binding of [2Fe-2S] clusters generally regulate Fur’s DNA binding ability, which is also associated with intracellular iron levels. Thus, it is anticipated that the ability of cluster-bound Fur to bind to DNA will vary depending on intracellular iron levels and other conditions.
Under conditions of iron hyperaccumulation, holo-Fur binds to the repressor site to inhibit further iron uptake. However, the binding affinity of Fur with Zn2+ at the S1 site is nearly 50 to 100 times stronger than that of Fur binding to repressor sites [45]. This disparity is cautiously thought to overcome the affinity difference due to the constant remodeling of the protein at the sensory site, potentially creating an artifact of the condition.

5. Structural Transition of Fur According to Its Metal Binding State and DNA Binding

Fur proteins are generally dimers in solution. However, Fur proteins can exist in a dynamic oligomeric state that varies depending on factors such as metal binding, DNA interactions, and protein-protein interactions. Some Fur proteins, such as those from P. aeruginosa, F. tularensis, and Legionella pneumophila, can form stable tetramers in solution [53,54]. Tetramer dissociation may be required to generate dimers capable of binding DNA [55]. From a structural perspective, while sometimes crystal packing can make the dimer of dimer form appear to exist in solution, its physiological significance may be ambiguous.
Fur proteins bind to specific DNA sequences known as the “Fur box” or “Fur-binding sites.” The general architecture of Fur boxes is conserved across bacteria, enabling Fur proteins to recognize and bind to these regulatory regions to control gene expression related to metal homeostasis and other cellular processes [56]. In E. coli, the Fur box is a 19 base pair (bp) consensus palindromic sequence (5′-GATAATGATAATCATTATC-3′) overlapping the -35 and -10 sites at the promoters of Fur-repressed genes [56,57,58]. It was proposed that the 19 bp inverted repeat consists of three GATAAT hexamers in a head-to-tail (6-6-1-6) orientation [59]. However, subsequent studies utilizing Bacillus subtilis Fur have demonstrated that the sequence can be viewed as two overlapping 15 base pair (7-1-7) inverted repeats [60].
When bound to DNA, the complex involving Fur proteins can consist of a single dimer, a dimer-of-dimers, or even an extended array of bound proteins. This DNA-bound complex is essential for Fur to exert its transcriptional regulatory functions by interacting with specific DNA sequences. In the case of the M. gryphiswaldense Fur-DNA complex, structures were determined in two forms of Fur [36] (Figure 4). One form is a dimer of Fur bound to the Fe2+ transporter protein feoAB1 operator, which contains the 7-1-7 consensus site. The other is two dimers of Fur bound to the E. coli (or P. aeruginosa) Fur box, which has two overlapping 7-1-7 consensus sites.
DNase I foot printing experiments highlight the different regulatory functions of H. pylori Fur in its dimeric and tetrameric (a dimer-of-dimer) forms. Under iron-deficient conditions, apo-Fur predominantly exists as a dimer, binds to a Fur operator named fOPII, but fails to activate. Under iron-sufficient conditions, Fur mainly exists as dimers with some tetramers, allowing Fur tetramers to bind to fOPII and induce activation. In conditions of excess iron, Fur predominantly forms tetramers, which bind to fOPIII and induce repression [61]. These different oligomeric states play a crucial role in the regulatory functions of Fur proteins in controlling metal homeostasis and gene expression in bacterial cells.
The Fur proteins exhibited conformational changes in accordance with the binding of metal ions (Figure 4). M. gryphiswaldense Fur underwent conformational changes of N-terminal DBDs and hinges upon the binding of two Mn2+ ions to sites S2 and S3. This binding resulted in the dimeric DD holding the DBD in a state that is optimal for binding the target DNA. Upon DNA binding, there is no significant structural rearrangement compared to metal-bound Fur [36].
With regard to C. jejuni Fur, two kinds of metal ion-bound structures were identified in the dimeric state. One of the identified structures involves the binding of two metal ions per Fur monomer to the sites S1 and S3 [32], while the other involves the binding of a single metal ion per Fur monomer to the S1 site [33]. Although additional binding of metal to site S3 did not result in significant structural alterations, it was demonstrated that the DBD can undergo asymmetric reorganization when the S3 site lacks metalation.

6. Fur Proteins Targeted Drug Discovery

Fur proteins are transcriptional regulators of genes involved in iron homeostasis, which is crucial for bacterial survival during infection [62]. Given that Fur regulates the production and uptake of siderophores, which chelate iron, Fur could be considered a therapeutic target, especially in conjunction with strategies to disrupt siderophore action [63]. Fur proteins have demonstrated various roles [20], including those related to virulence and colonization [64,65], quorum sensing [66], type III secretion [67], resistance to oxidative stress [68], and pH homeostasis [69]. Inactivation of the fur gene in various bacterial pathogens has been shown to decrease virulence in animal infection models [64,65,68,70,71]. One of the novel strategies for developing treatments for infectious diseases is the modulation of bacterial virulence. Consequently, targeting Fur, which is absent in eukaryotes, represents a novel approach to combating bacterial infections.
Several studies have focused on developing antibacterial agents targeting Fur. Specifically, anti-Fur peptide aptamers have been employed to target E. coli Fur. Using a yeast two-hybrid assay, a 20-million peptide library was screened to discover E. coli Fur inhibitors. As a result, four peptide aptamers, denoted F1 to F4, were selected for interacting with E. coli Fur [72]. To reduce the size of the aptamers, 13 amino acid-long linear peptides pF1 to pF4, corresponding to the variable loops of peptide aptamers F1 to F4, were investigated. Cissé et al. utilized the first anti-Fur linear peptide, pF1, and investigated the interaction between pF1 variations and E. coli in silico and in vitro experiments [73]. A more recent study by Mathieu et al. reported the inhibitory properties of pF1 to pF4, which prevent the binding of DNA to Fur [74]. In particular, pF2 showed a submicromolar dissociation constant (≤0.49 ± 0.10 μM), indicating its superior inhibitory capacity with E. coli Fur compared to pF1. The model structure of anti-Fur peptide inhibitors with the E. coli Fur complex obtained by docking exhibited the peptide inhibitors aligned through the valley of the V-shaped FUR dimer. Tyr56, Arg70, and Lys77 of E. coli Fur, conserved through homologs, were important in the DNA-binding structure of homologs (P. aeruginosa Fur-Fur box DNA, E. coli Fur-33mer DNA, M. gryphiswaldense Fur-feoAB1 DNA). In the docking model of the peptide inhibitor of E. coli Fur, these residues were shown to be involved.
To enhance the activity and cell permeability of peptide inhibitors, methods such as the α-helix stapling technique can be utilized to alter the α-helical content of linear peptides [75]. While this may not be applicable in all cases, optimization through stapling modifications has been reported to dramatically reduce minimum inhibitory concentration values, among other efficient applications [76,77]. Moreover, to optimize the interaction between Fur and potential inhibitors, employing thermodynamic profiling can yield improved results [78]. The thermodynamics of binding are influenced by multiple factors, including hydrogen bonding, hydrophobic interactions, desolvation, residual mobility, dynamics, and the local water structure. By utilizing thermodynamic profiling, we can consider these various elements to optimize a drug candidate to have desirable physicochemical properties.
Targeting Fur proteins in antibacterial drug discovery could involve exploring Fur homologs across species and designing advanced and selective Fur inhibitors using existing structures and computational techniques, including modeling and virtual screening. Additionally, evaluating the identified Fur inhibitors in combination therapies would be considered. These efforts could lead to innovative treatments and contribute significantly to addressing antibiotic resistance.

7. Conclusions

The Fur proteins serve as central regulators of iron homeostasis and metabolic processes in bacteria. Of the three metal-binding sites, S2 and S3 are highly conserved among bacteria, while S1 is absent in some species that lack the necessary cysteine residues. These differences may reflect variations in DNA binding among homologs and adaptations to specific environmental conditions. The role of Fur as a ferric uptake regulator, essential for bacterial survival, and its absence in eukaryotic cells make it an attractive drug target.
Consequently, peptide inhibitors have been explored to interfere with the DNA binding activity of Fur, utilizing peptide aptamers. In addition to peptide aptamers, peptide mimetic molecules or small molecules may also be employed in inhibitor development. Apart from, direct inhibitors of Fur-DNA binding, Fur can be considered a therapeutic target in conjunction with strategies to disrupt siderophore action. This approach is viable because Fur regulates siderophore production and uptake, as well as controlling the expression of genes that remove excess iron or directly absorb iron from heme.
Structural and functional studies of bacterial Fur proteins will provide critical information for the development of antibiotics, thereby contributing to the creation of effective drugs to overcome antibiotic resistance.

Author Contributions

Conceptualization, S.-M.K., H.-S.K. and D.-H.K.; software, S.-M.K. and D.-H.K.; validation, S.-M.K. and D.-H.K.; formal analysis, S.-M.K., H.-S.K. and D.-H.K.; investigation, S.-M.K., H.-S.K., W.-H.C., K.-T.K. and D.-H.K.; writing—original draft preparation, S.-M.K., H.-S.K. and D.-H.K.; writing—review and editing, S.-M.K., H.-S.K., W.-H.C., K.-T.K. and D.-H.K.; visualization, S.-M.K. and D.-H.K.; supervision, D.-H.K.; project administration, D.-H.K.; funding acquisition, S.-M.K. and D.-H.K. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Duksung Women’s University New Faculty Research Grants 2022. This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2022R1C1C1006354).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Weinberg, E.D. Iron availability and infection. Biochim. Biophys. Acta 2009, 1790, 600–605. [Google Scholar] [CrossRef] [PubMed]
  2. Archibald, F. Lactobacillus plantarum, an organism not requiring iron. FEMS Microbiol. Lett. 1983, 19, 29–32. [Google Scholar] [CrossRef]
  3. Posey, J.E.; Gherardini, F.C. Lack of a role for iron in the Lyme disease pathogen. Science 2000, 288, 1651–1653. [Google Scholar] [CrossRef] [PubMed]
  4. Frey, P.A.; Reed, G.H. The ubiquity of iron. ACS Chem. Biol. 2012, 7, 1477–1481. [Google Scholar] [CrossRef] [PubMed]
  5. Dian, C.; Vitale, S.; Leonard, G.A.; Bahlawane, C.; Fauquant, C.; Leduc, D.; Muller, C.; de Reuse, H.; Michaud-Soret, I.; Terradot, L. The structure of the Helicobacter pylori ferric uptake regulator Fur reveals three functional metal binding sites. Mol. Microbiol. 2011, 79, 1260–1275. [Google Scholar] [CrossRef] [PubMed]
  6. Methe, B.A.; Webster, J.; Nevin, K.; Butler, J.; Lovley, D.R. DNA microarray analysis of nitrogen fixation and Fe(III) reduction in Geobacter sulfurreducens. Appl. Environ. Microbiol. 2005, 71, 2530–2538. [Google Scholar] [CrossRef] [PubMed]
  7. Dancis, A.; Roman, D.G.; Anderson, G.J.; Hinnebusch, A.G.; Klausner, R.D. Ferric reductase of Saccharomyces cerevisiae: Molecular characterization, role in iron uptake, and transcriptional control by iron. Proc. Natl. Acad. Sci. USA 1992, 89, 3869–3873. [Google Scholar] [CrossRef] [PubMed]
  8. Thompson, D.K.; Beliaev, A.S.; Giometti, C.S.; Tollaksen, S.L.; Khare, T.; Lies, D.P.; Nealson, K.H.; Lim, H.; Yates, J., III; Brandt, C.C.; et al. Transcriptional and proteomic analysis of a ferric uptake regulator (fur) mutant of Shewanella oneidensis: Possible involvement of fur in energy metabolism, transcriptional regulation, and oxidative stress. Appl. Environ. Microbiol. 2002, 68, 881–892. [Google Scholar] [CrossRef] [PubMed]
  9. Kehrer, J.P. The Haber-Weiss reaction and mechanisms of toxicity. Toxicology 2000, 149, 43–50. [Google Scholar] [CrossRef]
  10. Kaushik, M.S.; Singh, P.; Tiwari, B.; Mishra, A.K. Ferric Uptake Regulator (FUR) protein: Properties and implications in cyanobacteria. Ann. Microbiol. 2016, 66, 61–75. [Google Scholar] [CrossRef]
  11. Lee, J.W.; Helmann, J.D. Functional specialization within the Fur family of metalloregulators. Biometals 2007, 20, 485–499. [Google Scholar] [CrossRef] [PubMed]
  12. Fillat, M.F. The FUR (ferric uptake regulator) superfamily: Diversity and versatility of key transcriptional regulators. Arch. Biochem. Biophys. 2014, 546, 41–52. [Google Scholar] [CrossRef] [PubMed]
  13. Hantke, K. Regulation of ferric iron transport in Escherichia coli K12: Isolation of a constitutive mutant. Mol. Gen. Genet. 1981, 182, 288–292. [Google Scholar] [CrossRef] [PubMed]
  14. Bagg, A.; Neilands, J.B. Ferric uptake regulation protein acts as a repressor, employing iron(II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry 1987, 26, 5471–5477. [Google Scholar] [CrossRef] [PubMed]
  15. Sevilla, E.; Bes, M.T.; Peleato, M.L.; Fillat, M.F. Fur-like proteins: Beyond the ferric uptake regulator (Fur) paralog. Arch. Biochem. Biophys. 2021, 701, 108770. [Google Scholar] [CrossRef] [PubMed]
  16. Pinochet-Barros, A.; Helmann, J.D. Redox Sensing by Fe2+ in Bacterial Fur Family Metalloregulators. Antioxid. Redox Signal 2018, 29, 1858–1871. [Google Scholar] [CrossRef] [PubMed]
  17. Troxell, B.; Hassan, H.M. Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria. Front. Cell. Infect. Microbiol. 2013, 3, 59. [Google Scholar] [CrossRef] [PubMed]
  18. Griggs, D.W.; Konisky, J. Mechanism for iron-regulated transcription of the Escherichia coli cir gene: Metal-dependent binding of fur protein to the promoters. J. Bacteriol. 1989, 171, 1048–1054. [Google Scholar] [CrossRef] [PubMed]
  19. Choi, S.S.; Chivers, P.T.; Berg, D.E. Point mutations in Helicobacter pylori’s fur regulatory gene that alter resistance to metronidazole, a prodrug activated by chemical reduction. PLoS ONE 2011, 6, e18236. [Google Scholar] [CrossRef]
  20. Carpenter, B.M.; Whitmire, J.M.; Merrell, D.S. This is not your mother’s repressor: The complex role of fur in pathogenesis. Infect. Immun. 2009, 77, 2590–2601. [Google Scholar] [CrossRef]
  21. Carpenter, B.M.; Gilbreath, J.J.; Pich, O.Q.; McKelvey, A.M.; Maynard, E.L.; Li, Z.Z.; Merrell, D.S. Identification and characterization of novel Helicobacter pylori apo-fur-regulated target genes. J. Bacteriol. 2013, 195, 5526–5539. [Google Scholar] [CrossRef] [PubMed]
  22. Delany, I.; Pacheco Ana Beatriz, F.; Spohn, G.; Rappuoli, R.; Scarlato, V. Iron-Dependent Transcription of the frpB Gene of Helicobacter pylori Is Controlled by the Fur Repressor Protein. J. Bacteriol. 2001, 183, 4932–4937. [Google Scholar] [CrossRef] [PubMed]
  23. Gilbreath, J.J.; West, A.L.; Pich, O.Q.; Carpenter, B.M.; Michel, S.; Merrell, D.S. Fur activates expression of the 2-oxoglutarate oxidoreductase genes (oorDABC) in Helicobacter pylori. J. Bacteriol. 2012, 194, 6490–6497. [Google Scholar] [CrossRef] [PubMed]
  24. Alamuri, P.; Mehta, N.; Burk, A.; Maier, R.J. Regulation of the Helicobacter pylori Fe-S cluster synthesis protein NifS by iron, oxidative stress conditions, and fur. J. Bacteriol. 2006, 188, 5325–5330. [Google Scholar] [CrossRef]
  25. Pich, O.Q.; Carpenter, B.M.; Gilbreath, J.J.; Merrell, D.S. Detailed analysis of Helicobacter pylori Fur-regulated promoters reveals a Fur box core sequence and novel Fur-regulated genes. Mol. Microbiol. 2012, 84, 921–941. [Google Scholar] [CrossRef] [PubMed]
  26. Bereswill, S.; Greiner, S.; van Vliet, A.H.; Waidner, B.; Fassbinder, F.; Schiltz, E.; Kusters, J.G.; Kist, M. Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J. Bacteriol. 2000, 182, 5948–5953. [Google Scholar] [CrossRef] [PubMed]
  27. Ernst, F.D.; Homuth, G.; Stoof, J.; Mader, U.; Waidner, B.; Kuipers, E.J.; Kist, M.; Kusters, J.G.; Bereswill, S.; van Vliet, A.H. Iron-responsive regulation of the Helicobacter pylori iron-cofactored superoxide dismutase SodB is mediated by Fur. J. Bacteriol. 2005, 187, 3687–3692. [Google Scholar] [CrossRef] [PubMed]
  28. Delany, I.; Spohn, G.; Rappuoli, R.; Scarlato, V. An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori. Mol. Microbiol. 2003, 50, 1329–1338. [Google Scholar] [CrossRef] [PubMed]
  29. Clark-Curtiss, J.E.; Curtiss, R., III. Salmonella Vaccines: Conduits for Protective Antigens. J. Immunol. 2018, 200, 39–48. [Google Scholar] [CrossRef]
  30. Swain, B.; Powell, C.T.; Curtiss, R., III. Pathogenicity and immunogenicity of Edwardsiella piscicida ferric uptake regulator (fur) mutations in zebrafish. Fish Shellfish. Immunol. 2020, 107, 497–510. [Google Scholar] [CrossRef]
  31. Seo, S.W.; Kim, D.; Latif, H.; O’Brien, E.J.; Szubin, R.; Palsson, B.O. Deciphering Fur transcriptional regulatory network highlights its complex role beyond iron metabolism in Escherichia coli. Nat. Commun. 2014, 5, 4910. [Google Scholar] [CrossRef] [PubMed]
  32. Butcher, J.; Sarvan, S.; Brunzelle, J.S.; Couture, J.F.; Stintzi, A. Structure and regulon of Campylobacter jejuni ferric uptake regulator Fur define apo-Fur regulation. Proc. Natl. Acad. Sci. USA 2012, 109, 10047–10052. [Google Scholar] [CrossRef] [PubMed]
  33. Sarvan, S.; Charih, F.; Askoura, M.; Butcher, J.; Brunzelle, J.S.; Stintzi, A.; Couture, J.F. Functional insights into the interplay between DNA interaction and metal coordination in ferric uptake regulators. Sci. Rep. 2018, 8, 7140. [Google Scholar] [CrossRef] [PubMed]
  34. Pecqueur, L.; D’Autreaux, B.; Dupuy, J.; Nicolet, Y.; Jacquamet, L.; Brutscher, B.; Michaud-Soret, I.; Bersch, B. Structural changes of Escherichia coli ferric uptake regulator during metal-dependent dimerization and activation explored by NMR and X-ray crystallography. J. Biol. Chem. 2006, 281, 21286–21295. [Google Scholar] [CrossRef]
  35. Perard, J.; Nader, S.; Levert, M.; Arnaud, L.; Carpentier, P.; Siebert, C.; Blanquet, F.; Cavazza, C.; Renesto, P.; Schneider, D.; et al. Structural and functional studies of the metalloregulator Fur identify a promoter-binding mechanism and its role in Francisella tularensis virulence. Commun. Biol. 2018, 1, 93. [Google Scholar] [CrossRef] [PubMed]
  36. Deng, Z.Q.; Wang, Q.; Liu, Z.; Zhang, M.F.; Machado, A.C.D.; Chiu, T.P.; Feng, C.; Zhang, Q.; Yu, L.; Qi, L.; et al. Mechanistic insights into metal ion activation and operator recognition by the ferric uptake regulator. Nat. Commun. 2015, 6, 7642. [Google Scholar] [CrossRef] [PubMed]
  37. Pohl, E.; Haller, J.C.; Mijovilovich, A.; Meyer-Klaucke, W.; Garman, E.; Vasil, M.L. Architecture of a protein central to iron homeostasis: Crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol. Microbiol. 2003, 47, 903–915. [Google Scholar] [CrossRef]
  38. Sheikh, M.A.; Taylor, G.L. Crystal structure of the Vibrio cholerae ferric uptake regulator (Fur) reveals insights into metal co-ordination. Mol. Microbiol. 2009, 72, 1208–1220. [Google Scholar] [CrossRef] [PubMed]
  39. Coy, M.; Neilands, J.B. Structural dynamics and functional domains of the fur protein. Biochemistry 1991, 30, 8201–8210. [Google Scholar] [CrossRef]
  40. Stojiljkovic, I.; Hantke, K. Functional domains of the Escherichia coli ferric uptake regulator protein (Fur). Mol. Gen. Genet. 1995, 247, 199–205. [Google Scholar] [CrossRef]
  41. Fontenot, C.R.; Tasnim, H.; Valdes, K.A.; Popescu, C.V.; Ding, H. 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. [Google Scholar] [CrossRef]
  42. Mills, S.A.; Marletta, M.A. Metal binding characteristics and role of iron oxidation in the ferric uptake regulator from Escherichia coli. Biochemistry 2005, 44, 13553–13559. [Google Scholar] [CrossRef] [PubMed]
  43. Hohle, T.H.; O’Brian, M.R. Metal-specific control of gene expression mediated by Bradyrhizobium japonicum Mur and Escherichia coli Fur is determined by the cellular context. Mol. Microbiol. 2016, 101, 152–166. [Google Scholar] [CrossRef]
  44. Brawley, H.N.; Kreinbrink, A.C.; Hierholzer, J.D.; Vali, S.W.; Lindahl, P.A. Labile Iron Pool of Isolated Escherichia coli Cytosol Likely Includes Fe-ATP and Fe-Citrate but not Fe-Glutathione or Aqueous Fe. J. Am. Chem. Soc. 2023, 145, 2104–2117. [Google Scholar] [CrossRef] [PubMed]
  45. Fontenot, C.R.; Ding, H. Ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to regulate intracellular iron homeostasis in Escherichia coli. J. Biol. Chem. 2023, 299, 104748. [Google Scholar] [CrossRef] [PubMed]
  46. Shin, J.H.; Jung, H.J.; An, Y.J.; Cho, Y.B.; Cha, S.S.; Roe, J.H. Graded expression of zinc-responsive genes through two regulatory zinc-binding sites in Zur. Proc. Natl. Acad. Sci. USA 2011, 108, 5045–5050. [Google Scholar] [CrossRef] [PubMed]
  47. Lucarelli, D.; Russo, S.; Garman, E.; Milano, A.; Meyer-Klaucke, W.; Pohl, E. Crystal structure and function of the zinc uptake regulator FurB from Mycobacterium tuberculosis. J. Biol. Chem. 2007, 282, 9914–9922. [Google Scholar] [CrossRef] [PubMed]
  48. Madeira, F.; Pearce, M.; Tivey, A.R.N.; Basutkar, P.; Lee, J.; Edbali, O.; Madhusoodanan, N.; Kolesnikov, A.; Lopez, R. Search and sequence analysis tools services from EMBL-EBI in 2022. Nucleic Acids Res. 2022, 50, W276–W279. [Google Scholar] [CrossRef]
  49. Robert, X.; Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014, 42, W320–W324. [Google Scholar] [CrossRef] [PubMed]
  50. D’Autreaux, B.; Pecqueur, L.; Gonzalez de Peredo, A.; Diederix, R.E.; Caux-Thang, C.; Tabet, L.; Bersch, B.; Forest, E.; Michaud-Soret, I. Reversible redox- and zinc-dependent dimerization of the Escherichia coli fur protein. Biochemistry 2007, 46, 1329–1342. [Google Scholar] [CrossRef]
  51. Fontenot, C.R.; Ding, H. Ferric uptake regulators (Fur) from Vibrio cholerae and Helicobacter pylori bind a [2Fe-2S] cluster in response to elevation of intracellular free iron content. Biometals 2022, 35, 591–600. [Google Scholar] [CrossRef]
  52. Fontenot, C.R.; Ding, H. The C-terminal domain of the ferric uptake regulator (Fur) binds a [2Fe-2S] cluster to sense the intracellular free iron content in Escherichia coli. Biometals 2023, 36, 1285–1294. [Google Scholar] [CrossRef]
  53. Pérard, J.; Covès, J.; Castellan, M.; Solard, C.; Savard, M.; Miras, R.; Galop, S.; Signor, L.; Crouzy, S.; Michaud-Soret, I.; et al. Quaternary Structure of Fur Proteins, a New Subfamily of Tetrameric Proteins. Biochemistry 2016, 55, 1503–1515. [Google Scholar] [CrossRef] [PubMed]
  54. Nader, S.; Perard, J.; Carpentier, P.; Arnaud, L.; Crouzy, S.; Michaud-Soret, I. New insights into the tetrameric family of the Fur metalloregulators. Biometals 2019, 32, 501–519. [Google Scholar] [CrossRef]
  55. Steingard, C.H.; Helmann, J.D. Meddling with Metal Sensors: Fur-Family Proteins as Signaling Hubs. J. Bacteriol. 2023, 205, e0002223. [Google Scholar] [CrossRef] [PubMed]
  56. Escolar, L.; Perez-Martin, J.; de Lorenzo, V. Opening the iron box: Transcriptional metalloregulation by the Fur protein. J. Bacteriol. 1999, 181, 6223–6229. [Google Scholar] [CrossRef]
  57. Andrews, S.C.; Robinson, A.K.; Rodriguez-Quinones, F. Bacterial iron homeostasis. FEMS Microbiol. Rev. 2003, 27, 215–237. [Google Scholar] [CrossRef] [PubMed]
  58. de Lorenzo, V.; Wee, S.; Herrero, M.; Neilands, J.B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J. Bacteriol. 1987, 169, 2624–2630. [Google Scholar] [CrossRef]
  59. Escolar, L.; Perez-Martin, J.; de Lorenzo, V. Binding of the fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence. J. Mol. Biol. 1998, 283, 537–547. [Google Scholar] [CrossRef]
  60. Baichoo, N.; Helmann, J.D. Recognition of DNA by Fur: A reinterpretation of the Fur box consensus sequence. J. Bacteriol. 2002, 184, 5826–5832. [Google Scholar] [CrossRef]
  61. Roncarati, D.; Pelliciari, S.; Doniselli, N.; Maggi, S.; Vannini, A.; Valzania, L.; Mazzei, L.; Zambelli, B.; Rivetti, C.; Danielli, A. Metal-responsive promoter DNA compaction by the ferric uptake regulator. Nat. Commun. 2016, 7, 12593. [Google Scholar] [CrossRef] [PubMed]
  62. Hantke, K. Iron and metal regulation in bacteria. Curr. Opin. Microbiol. 2001, 4, 172–177. [Google Scholar] [CrossRef] [PubMed]
  63. Smith, D.J.; Lamont, I.L.; Anderson, G.J.; Reid, D.W. Targeting iron uptake to control Pseudomonas aeruginosa infections in cystic fibrosis. Eur. Respir. J. 2013, 42, 1723–1736. [Google Scholar] [CrossRef] [PubMed]
  64. Pich, O.Q.; Merrell, D.S. The ferric uptake regulator of Helicobacter pylori: A critical player in the battle for iron and colonization of the stomach. Future Microbiol. 2013, 8, 725–738. [Google Scholar] [CrossRef]
  65. Mey, A.R.; Wyckoff, E.E.; Kanukurthy, V.; Fisher, C.R.; Payne, S.M. Iron and fur regulation in Vibrio cholerae and the role of fur in virulence. Infect. Immun. 2005, 73, 8167–8178. [Google Scholar] [CrossRef] [PubMed]
  66. Oglesby, A.G.; Farrow, J.M., III; Lee, J.H.; Tomaras, A.P.; Greenberg, E.P.; Pesci, E.C.; Vasil, M.L. The influence of iron on Pseudomonas aeruginosa physiology: A regulatory link between iron and quorum sensing. J. Biol. Chem. 2008, 283, 15558–15567. [Google Scholar] [CrossRef]
  67. Ellermeier, J.R.; Slauch, J.M. Fur regulates expression of the Salmonella pathogenicity island 1 type III secretion system through HilD. J. Bacteriol. 2008, 190, 476–486. [Google Scholar] [CrossRef] [PubMed]
  68. Horsburgh, M.J.; Ingham, E.; Foster, S.J. In Staphylococcus aureus, Fur is an interactive regulator with PerR, contributes to virulence, and is necessary for oxidative stress resistance through positive regulation of catalase and iron homeostasis. J. Bacteriol. 2001, 183, 468–475. [Google Scholar] [CrossRef] [PubMed]
  69. Gancz, H.; Censini, S.; Merrell, D.S. Iron and pH homeostasis intersect at the level of fur regulation in the gastric pathogen. Infect. Immun. 2006, 74, 602–614. [Google Scholar] [CrossRef]
  70. Hassett, D.J.; Sokol, P.A.; Howell, M.L.; Ma, J.F.; Schweizer, H.T.; Ochsner, U.; Vasil, M.L. Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J. Bacteriol. 1996, 178, 3996–4003. [Google Scholar] [CrossRef]
  71. Huja, S.; Oren, Y.; Biran, D.; Meyer, S.; Dobrindt, U.; Bernhard, J.; Becher, D.; Hecker, M.; Sorek, R.; Ron, E.Z. Fur is the master regulator of the extraintestinal pathogenic Escherichia coli response to serum. mBio 2014, 5, e01460-14. [Google Scholar] [CrossRef] [PubMed]
  72. Abed, N.; Bickle, M.; Mari, B.; Schapira, M.; Sanjuan-Espana, R.; Robbe Sermesant, K.; Moncorge, O.; Mouradian-Garcia, S.; Barbry, P.; Rudkin, B.B.; et al. A comparative analysis of perturbations caused by a gene knock-out, a dominant negative allele, and a set of peptide aptamers. Mol. Cell. Proteom. 2007, 6, 2110–2121. [Google Scholar] [CrossRef] [PubMed]
  73. Cisse, C.; Mathieu, S.V.; Abeih, M.B.; Flanagan, L.; Vitale, S.; Catty, P.; Boturyn, D.; Michaud-Soret, I.; Crouzy, S. Inhibition of the ferric uptake regulator by peptides derived from anti-FUR peptide aptamers: Coupled theoretical and experimental approaches. ACS Chem. Biol. 2014, 9, 2779–2786. [Google Scholar] [CrossRef] [PubMed]
  74. Mathieu, S.; Cisse, C.; Vitale, S.; Ahmadova, A.; Degardin, M.; Perard, J.; Colas, P.; Miras, R.; Boturyn, D.; Coves, J.; et al. From Peptide Aptamers to Inhibitors of FUR, Bacterial Transcriptional Regulator of Iron Homeostasis and Virulence. ACS Chem. Biol. 2016, 11, 2519–2528. [Google Scholar] [CrossRef]
  75. Kim, Y.W.; Grossmann, T.N.; Verdine, G.L. Synthesis of all-hydrocarbon stapled alpha-helical peptides by ring-closing olefin metathesis. Nat. Protoc. 2011, 6, 761–771. [Google Scholar] [CrossRef] [PubMed]
  76. Kang, S.M.; Moon, H.; Han, S.W.; Kim, D.H.; Kim, B.M.; Lee, B.J. Structure-Based De Novo Design of Mycobacterium Tuberculosis VapC-Activating Stapled Peptides. ACS Chem. Biol. 2020, 15, 2493–2498. [Google Scholar] [CrossRef] [PubMed]
  77. Kang, S.M.; Moon, H.; Han, S.W.; Kim, B.W.; Kim, D.H.; Kim, B.M.; Lee, B.J. Toxin-Activating Stapled Peptides Discovered by Structural Analysis Were Identified as New Therapeutic Candidates That Trigger Antibacterial Activity against Mycobacterium tuberculosis in the Mycobacterium smegmatis Model. Microorganisms 2021, 9, 568. [Google Scholar] [CrossRef]
  78. Klebe, G. Applying thermodynamic profiling in lead finding and optimization. Nat. Rev. Drug Discov. 2015, 14, 95–110. [Google Scholar] [CrossRef]
Biomolecules 14 00981 g001
Figure 1. Iron-dependent transcription regulation by Fur. The concentration of Fe2+ affects the Fe-bound state of the Fur protein, which exists as Fe-Fur bound to the S2 site at high Fe2+ concentrations and as apo-Fur with Fe2+ removed from S2 at low Fe2+ concentrations. In H. pylori, Fur proteins can act in four ways: apo-Fur activation, apo-Fur repression, Fe-Fur (holo-Fur) activation, and Fe-Fur repression. Apo-Fur can repress the transcription of genes such as prf and sodB or be involved in the transcriptional activation of fur. Fe-Fur can repress the transcription of iron uptake-related genes such as frpB or activate the transcription of genes such as oorDABC, nifS, and cagA.
Figure 1. Iron-dependent transcription regulation by Fur. The concentration of Fe2+ affects the Fe-bound state of the Fur protein, which exists as Fe-Fur bound to the S2 site at high Fe2+ concentrations and as apo-Fur with Fe2+ removed from S2 at low Fe2+ concentrations. In H. pylori, Fur proteins can act in four ways: apo-Fur activation, apo-Fur repression, Fe-Fur (holo-Fur) activation, and Fe-Fur repression. Apo-Fur can repress the transcription of genes such as prf and sodB or be involved in the transcriptional activation of fur. Fe-Fur can repress the transcription of iron uptake-related genes such as frpB or activate the transcription of genes such as oorDABC, nifS, and cagA.
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Figure 2. Overall structure of Fur with three metal-binding sites. The structure of H. pylori Fur (PDB ID: 2XIG) was employed. Fur proteins typically exist as dimers, and the Fur protein monomer is divided into two domains: the N-terminal DBD, shown in green, and the C-terminal DD, shown in blue. The three metals (Zn2+ in this structure) present at the S1, S2, and S3 sites are shown in grey, red, and yellow, respectively. The detailed interactions between the metal and ligands at each site are presented in an enlarged view.
Figure 2. Overall structure of Fur with three metal-binding sites. The structure of H. pylori Fur (PDB ID: 2XIG) was employed. Fur proteins typically exist as dimers, and the Fur protein monomer is divided into two domains: the N-terminal DBD, shown in green, and the C-terminal DD, shown in blue. The three metals (Zn2+ in this structure) present at the S1, S2, and S3 sites are shown in grey, red, and yellow, respectively. The detailed interactions between the metal and ligands at each site are presented in an enlarged view.
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Figure 3. Sequence alignment of Fur proteins. The secondary structures are indicated at the top of the alignment. The highly conserved residues are highlighted in red boxes. The metal-binding ligands of sites S1, S2, and S3 are indicated by grey, red, and yellow boxes, respectively, below the residues. Multiple sequence alignment and visualization were conducted using Clustal Omega [48] and ESPript 3.0 [49], respectively.
Figure 3. Sequence alignment of Fur proteins. The secondary structures are indicated at the top of the alignment. The highly conserved residues are highlighted in red boxes. The metal-binding ligands of sites S1, S2, and S3 are indicated by grey, red, and yellow boxes, respectively, below the residues. Multiple sequence alignment and visualization were conducted using Clustal Omega [48] and ESPript 3.0 [49], respectively.
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Figure 4. Structural differences according to the ligand binding state. In M. gryphiswaldense Fur, a comparison of the apo- and holo-Fur structures reveals that the dimer in holo-Fur is more symmetrical. Metal binding to site S2 of Fur causes a conformational change in the DBD, facilitating DNA binding. Different conformations of the Fur protein can be adopted for DNA binding. The M. gryphiswaldense Fur protein was observed to bind to the feoAB1 operator with the 7-1-7 consensus site as a single dimer and to the Fur box, which contains two overlapping 7-1-7 consensus sequences, as two dimers.
Figure 4. Structural differences according to the ligand binding state. In M. gryphiswaldense Fur, a comparison of the apo- and holo-Fur structures reveals that the dimer in holo-Fur is more symmetrical. Metal binding to site S2 of Fur causes a conformational change in the DBD, facilitating DNA binding. Different conformations of the Fur protein can be adopted for DNA binding. The M. gryphiswaldense Fur protein was observed to bind to the feoAB1 operator with the 7-1-7 consensus site as a single dimer and to the Fur box, which contains two overlapping 7-1-7 consensus sequences, as two dimers.
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Table 1. Structural information of Fur proteins.
Table 1. Structural information of Fur proteins.
OrganismsPDB IDMetals per MonomerMetal Binding SiteBound DNAReferences
S1S2S3
C. jejuni4ETS2 Zn2+Zn2+ Zn2+ [32]
6D57Zn2+Zn2+ [33]
E. coli2FU4 [34]
F. tularensis5NHKFe2+, Zn2+Zn2+Fe2+ [35]
5NBCMn2+, Zn2+Zn2+Mn2+
H. pylori2XIG3 Zn2+Zn2+Zn2+Zn2+ [5]
M. gryphiswaldense4RAY- [36]
4RAZ2 Mn2+ Mn2+Mn2+
4RB32 Mn2+ Mn2+Mn2+feoAB1 operator
4RB12 Mn2+ Mn2+Mn2+Fur box
P. aeruginosa1MZB2 Zn2+ Zn2+Zn2+ [37]
V. cholerae2W572 Zn2+ Zn2+Zn2+ [38]
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Kang, S.-M.; Kang, H.-S.; Chung, W.-H.; Kang, K.-T.; Kim, D.-H. Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur). Biomolecules 2024, 14, 981. https://doi.org/10.3390/biom14080981

AMA Style

Kang S-M, Kang H-S, Chung W-H, Kang K-T, Kim D-H. Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur). Biomolecules. 2024; 14(8):981. https://doi.org/10.3390/biom14080981

Chicago/Turabian Style

Kang, Sung-Min, Hoon-Seok Kang, Woo-Hyun Chung, Kyu-Tae Kang, and Do-Hee Kim. 2024. "Structural Perspectives on Metal Dependent Roles of Ferric Uptake Regulator (Fur)" Biomolecules 14, no. 8: 981. https://doi.org/10.3390/biom14080981

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