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.
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).
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).
The generalized transport reaction catalyzed by FeoB is presumably:
Fe2+ (out) + energy (GTP hydrolysis) → Fe2+ (in)