9.A.8 The Ferrous Iron Uptake (FeoB) Family
The FeoB protein of E. coli is an integral membrane protein of 773 amino acyl residues which is predicted to span the membrane 8-13 times as α-helices (Kammler et al., 1993). Homologous proteins are encoded within the genomes of many bacteria and archaea. The E. coli protein possesses an N-terminal 300 amino acyl residue hydrophilic domain that bears at its N-terminus a regulatory ATP/GTP binding motif as well as an S domain. The N-terminal hydrophilic domain is homologous to prokaryotic and eukaryotic GTP binding proteins including the E. coli Era protein (P06616). GTP binding is required for efficient Fe2+ uptake, but GTP is hydrolyzed very slowly (Marlovits et al., 2002). The C-terminal transmembrane domain of FeoB catalyzes transport (Hantke, 2003; Hung et al. 2012). Transport is probably regulated by or energized by the N-terminal intramolecular G-protein-like domain. Based on x-ray crystallographic data, the G-doman transmits information to the transmembrane domain in a fashion possibly similar to energy transfer in ABC transporters (Köster et al., 2009). Biochemical analyses demonstrated that the GTPase activity of FeoB is activated by K+, which leads to a 20-fold acceleration in its hydrolysis rate (Ash et al. 2010). Analysis of the structure identified a conserved asparagine residue likely to be involved in K+ coordination, and mutation of this residue abolished K+-dependent activation. Ash et al. (2010) suggested that this, together with a second asparagine residue that is critical for the structure of the Switch I loop, allows K+-dependent activation in G proteins. The accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter. Iron uptake in eukaryotes has been reviewed (Sherman et al. 2018) as has that in prokaryotes (Sestok et al. 2018). Thus the mechanism of energy coupling to FeoB transport is an open question.
A FeoB homologue is present in Helicobacter pylori. This system takes up Fe2+ with high affinity (0.5 μM) in a process that is inhibited by FCCP, DCCD and vanadate, indicating that uptake is energized by ATP or GTP hydrolysis (Velayudhan et al., 2000). Fe3+ is first converted to Fe2+ by an extracytoplasmic Fe3+ reductase, and the resultant Fe2+ is taken up by FeoB. FeoB appears to provide the major pathway for Fe2+ uptake in H. pylori and C. perfringens, and it is essential for colonization of the murine gastric mucosa in H. pylori. A similar FeoB homologue in the spirochete Leptospira biflexa has been implicated in Fe2+ uptake (Louvel et al., 2005). A general protocol for the expression and purification of the intact transmembrane FeoBs from several bacteria has been described (Sestok et al. 2022).
Prokaryotic FeoB proteins are involved in G protein coupled Fe2+ transport. They are unique in that the G protein is directly tethered to the membrane domain. Guilfoyle et al., 2009 reported the structure of the soluble domain of FeoB, including the G protein domain, and its assembly into a trimer. Comparisons between nucleotide free and liganded structures reveal the closed and open state of a central cytoplasmic pore, respectively. In addition, these data provide the first observation of a conformational switch in the nucleotide-binding G5 motif, defining the structural basis for GDP release. From these results, structural parallels are drawn to eukaryotic G protein coupled membrane processes (Guilfoyle et al., 2009).
The Feo transport system consists of three proteins: FeoA, FeoB, and FeoC. The N-terminal domain (N-FeoB) has been shown to form a trimeric pore that may function as a Fe2+ gate. FeoC is a small winged-helix protein possessing four conserved cysteine residues with a consensus sequence that may provide binding sites for an [Fe-S]-cluster. Therefore, FeoC may be an [Fe-S]-cluster-dependent regulator that directly controls transcription of the feo operon. Hung et al. (2012) showed that Klebsiella pneumoniae FeoC (KpFeoC) forms a tight complex with the intracellular N-terminal domain of FeoB (KpNFeoB). The crystal structure of the complex revealed that KpFeoC binds to KpNFeoB between the switch II region of the G-protein domain and the effector S domain, and that the long KpFeoC W1 loop lies above the KpNFeoB nucleotide-binding site. These interactions suggest that KpFeoC modulates guanine nucleotide-mediated signal transduction. Binding of KpFeoC disrupts pore formation by interfering with KpNFeoB trimerization. Thus, KpFeoC may play a crucial role in regulating Fe2+ transport as well as gene regulation. FeoA is a 75aa protein homologous to the N-terminus of FeoB2 of Porphyromonas gingivalis (TC#9.A.8.1.6) and some similarity to an internal hydrophilic segment of the RND heavy metal porter, CzcA of Myxococcus xanthus (TC#2.A.6.1.7).
In Vibrio cholerae the feo operon consists of three genes, feoABC. feoB encodes an 83 kDa protein with an amino terminal GTPase domain and a carboxy terminal domain predicted to be embedded in the inner membrane. In V. cholerae, FeoA and FeoC, as well as the more highly conserved FeoB, are all required for iron acquisition (Weaver et al. 2013). FeoC interacts with the cytoplasmic domain of FeoB, and two conserved amino acids in FeoC were found to be necessary for the interaction with FeoB.
FeoB normally consists of a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. NFeoB has two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe2+ transport. The structure of the cytoplasmic domain of FeoB from Gallionella capsiferriformans has been determined (Deshpande et al. 2013). The G. capsiferriformans NFeoB structure does not contain a helical domain, and the crystal structures of both the apo and GDP-bound protein reveals a domain-swapped dimer. I
Insertional inactivation of feoB in C. perfringens yielded altered growth properties and a markedly reduced total iron and manganese content compared to the wild type. Thus, under anaerobic conditions, FeoB is the major protein required for iron uptake into the cell and it may play an important role in the pathogenesis of C. perfringens infections (Awad et al. 2016).
Bacterial FeoBs possess either GTPase or an NTPase with substrate promiscuity. This difference in nucleotide preference alters cellular requirements for monovalent and divalent cations. While the hydrolytic activity of the GTP-dependent FeoBs was stimulated by potassium, the action of the NTP-dependent FeoBs was not. Mutation of Asn11, having a role in potassium-dependent GTP hydrolysis, changed nucleotide specificity of the NTP-dependent FeoB, resulting in loss of ATPase activity (Shin et al. 2020).
Feo transporters are the most widespread systems for ferrous iron uptake in bacteria and are critical for virulence in some species. The canonical architecture of Feo consists of a large transmembrane nucleoside triphosphatase (NTPase) protein, FeoB, and two small accessory cytoplasmic proteins, FeoA and FeoC. Gómez-Garzón et al. 2022 conducted comparative analyses of Feo protein sequences. They found that FeoC, while absent in most lineages, is largely present in the γ-proteobacteria although its sequence is poorly conserved. FeoC may couple FeoB NTPase activity with pore opening and was an ancestral element that has been dispensed with through mutations in FeoA and FeoB. FeoC-independent mutants of the Vibrio cholerae Feo system were isolated, and the Shewanella oneidensis FeoAB does not require FeoC; thus, the Vibrio FeoC sequences may resemble transitional forms on an evolutionary pathway toward FeoC-independent transporters. FeoC may have different functions in different species that retain this protein, and the loss of FeoC may be promoted by mutations in FeoA or by the fusion of FeoA and FeoB (Gómez-Garzón et al. 2022).
The generalized transport reaction catalyzed by FeoB is presumably:
Fe2+ (out) + energy (GTP hydrolysis) → Fe2+ (in)
Ferrous iron uptake system, FeoAB. FeoB is the main transporter while FeoC is a transcriptional regulator. FeoA has a Src-Homology (SH3) domain (a β-barrel with two overlying α-helices) plus two extra α-helices not usually found in SH3 domains. FeoA may interact with the cytoplasmic domain of the conserved core portion of the FeoB transmembrane region, but may not function as a GTPase activator as previously proposed (Lau et al. 2012).
FeoAB of E. coli
FeoABC Fe2+ transporter (Hung et al., 2012). FeoA is a 75 aa protein homologous to the N-terminus of FeoB2 of Porphyromonas gingivalis (TC#9.A.8.1.6) and with some sequence similarity to an internal hydrophilic segment of the RND heavy metal porter, CzcA of Myxococcus xanthus (TC#2.A.6.1.7). Linkous et al. 2019 have determined the crystal structure of FeoA from Klebsiella pneumoniae (KpFeoA). The structure reveals an SH3-like domain that mediates interactions between neighboring polypeptides via hydrophobic intercalations into a Leu-rich surface ridge. Using docking of a small peptide corresponding to a postulated FeoB partner binding site, they demonstrated that KpFeoA can assume both "open" and "closed" conformations, controlled by binding at this Leu-rich ridge. They propose a model in which a "C-shaped" clamp along the FeoA surface mediates interactions with its partner protein, FeoB (Linkous et al. 2019).
FeoABC of Klebsiella pneumoniae
FeoB of 595 aas. The structure of the N-terminal GTPase domain has been determined by NMR (Deshpande et al. 2013). GTP hydrolysis has been reported to be required for transport activity.
FeoB of Gallionella capsiferriformans (strain ES-2) (Gallionella ferruginea capsiferriformans (strain ES-2))
Iron transporter, FeoB, of 751 aas and 9 putative TMSs with a C-terminal GTPase domain. Plays a key role in iron uptake and virulence. The crystal structure of the N-terminal cytosolic domain (NFeoB) is known (Petermann et al. 2010). The strucuture reveals a monomeric protein comprised of two separate sub-domains with GTPase and guanine-nucleotide dissociation inhibitor (GDI) functions, respectively. The GDI domain was found to display a novel fold, whereas the GTPase domain resembled that of known G domains. The crystalized protein was in the rarely observed nucleotide-free state.
FeoB of Legionella pneumophila, the causative agent of Legionnaires' disease
FepB of 827 aas and 9 TMSs. Fe2+ uptake system, probably driven by GTP (Veeranagouda et al. 2014).
FeoB of Bacteroides fragilis
Ferrous iron ion uptake transporter of 669 aas and 10 TMSs in a 5 + 5 arrangement (C-terminal, with an N-terminal GTPase). Under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the C. perfringens pathogenesis (Awad et al. 2016).
FeoB of Clostridium perfringens
FeoB of 766 aas and 10 TMSs, a potential GTP hydrolysis-driven active transporter or GTP-activated iron uptake channel. The membrane domain of the trimeric FeoB forms a central pore lined by highly conserved cysteine residues. This pore aligns with a central pore in the N-terminal GTPase domain (G-domain) which is lined by aspartyl residues. Biochemical analyses revealed a putative iron sensor domain that could connect GTP binding/hydrolysis to the opening of the pore. Thus, FeoB may be a GTP-gated channel or GTP hydrolysis-driven primary active transporter (Seyedmohammad et al. 2016).
FeoB of Pseudomonas aeruginosa
FeoA2/B2 of 78 and 788 aas, respectively.
FeoA2B2 of Magnetospirillum gryphiswaldense MSR-1 v2
Putative FeoB homologue of 440 aas and 11 TMSs in a 3 + 3 + 5 TMS arrangement. It may function with a putative GTP-binding protein EngB. This proposed system is lacking a five-helix helical domain, which however, other proteins in this family also lack.
FeoB homologue + GTPase of Candidatus Thorarchaeota archaeon
FeoABC ferrous iron (Fe2+) uptake system. All three subunits are required for activity (Gómez-Garzón et al. 2022). Subunits FeoA and FeoC are the same size and show very similar hydrophathy plots. Many Feo transport systems lack a FeoC subunit (e.g., see 9.A.8.1.19), and mutations in FeoA can eliminate the need for FeoC (see family description; Gómez-Garzón et al. 2022).
FeoABC of Vibrio cholerae serotype O1
FeoA, 76 aas and possibly 1 central TMS, C3LP28
FeoB, 758 aas and 9 or 10 TMSs, C3LP27
FeoC, 76 aas and possibly 1 central TMS, C3LP26
FeoBA ferrous iron uptake system. There is no FeoC in this systems (Gómez-Garzón et al. 2022).
FeoBA of Shewanella oneidensis (strain MR-1)
FeoB, 764 aas and 9 or 10 TMSs, Q8EG28
FeoA, 76 aas and 0 TMSs, Q8EG29
FeoC-like transcriptional regulator of 95 aas. The N-terminal domain is the FeoC domain.
FeoC-like Tx regulator of Microbispora oryzae
Manganous ion (Mn2+) uptake system, FeoB2 (Dashper et al., 2005)
FeoB2 of Porphyromonas gingivalis (AAQ66370)
FeoAB of 704 and 84 aas, respectively (Uebe and Schüler 2016).
FeoAB of Magnetospirillum gryphiswaldense
The putative Fe2+ transport protein B, FeoB (COG3366 family)
FeoB of Archaeoglobus fulgidus (O29993)