FepA

FepA is an integral bacterial outer membrane porin protein, which is involved in the active transport of iron from the extracellular space, into the periplasm of Gram-negative bacteria. A Gram-negative bacterium secretes iron-binding proteins called siderophores, which bind strongly to ferric ions. The FepA receptor is present at the outer membrane and binds to ferric siderophores, and actively transport it into the cell. FepA has also been shown to transport vitamin B12, and colicins B and D as well.^[1] This protein belongs to family of ligand-gated protein channels.

Because no energy is directly available to the outer membrane, FepA has been studied to determine the mechanism by which energy is brought to the outer membrane to drive the transport. It was found that the energy to drive the active transport originates from the proton motive force (electrochemical gradient) generated in the inner membrane complex TonB–ExbB–ExbD, and this force is relayed physically to FepA through the TonB subunit.

Structure

The ferric ion pictured is bound by 3 catechol groups, as happens with the molecule enterobactin

Using X-ray crystallography the structure of FepA was found to be a 724-residue 22-stranded β-barrel. The barrel contains loops that act as high-affinity and high-specificity ligand-binding sites outside the cell. The N-terminus forms a smaller barrel domain inside the hydrophilic barrel pore, effectively closing the pore; from studies of FhuA, a similar TonB-dependent outer membrane transporter, the interaction of the N-terminus domain to the inner walls of the pore is thought to be strengthened by nine salt-bridges and over 60 hydrogen bonds. The N-terminus also has a further two extracellular loops in the pore, which are thought to aid in the signal transduction between ligand-binding and TonB-mediated transport, though the precise mechanism is not clear. Residues 12 to 18 of the N-terminus domain of FepA comprises a region called the TonB box, which includes at least a proline and glycine residue. The TonB box interacts with a proline-rich motif consisting of a conserved Gln160 residue and the C-terminus 48 residues. When they interact, the conformation of the N-terminal domain is changed so as to open the pore. In vivo crosslinking confirms that this interaction is physical. It is however energetically nonsensical to remove the whole of the N-terminal domain for translocation, because this requires the breakage of the salt bridges and numerous hydrogen bonds, and so it is assumed that the displace is only slight, just large enough for transport of FeEnt. The role of the N-terminus is revealed by using a deletion mutation of the N-terminal plug; the protein was still able to be inserted into the membrane, but acts as a non-selective pore for larger molecules, exhibited by increased permeability of the cell to maltotetraose, maltopentaose, ferrichrome, as well as several antibiotics including albomycin, vancomycin and bacitracin. However, this have to be treated with caution, as the conformation of the barrel may change in the absence of the N-terminal plug.

Enterobactin is a cyclic tri-ester of 2,3-dihydroxybenzoylserine with a molecular mass of 719 Da. It binds ferric ions using six oxygens from three catechol groups, giving an overall charge of −3. Like the binding catechol, enterobactin is thought to also have a three-fold symmetry dissecting the metal centre.^[2]

Function

Iron is not usually readily available in the environment this group of bacteria find themselves in. However iron is essential in sustaining life due to its role in co-enzymes of respiration and DNA synthesis, so bacteria must adapt to have a mechanism for intake of iron. Because Fe³⁺ has a very low solubility, most of the Fe³⁺ ions in the bacteria’s surrounding environment (e.g. soil) exist as iron oxides or hydroxides, and so the number of free Fe³⁺ is low. Therefore, microbes have evolved to secret siderophores, Fe³⁺-binding peptides, into the surroundings and then actively transport the Fe³⁺-complex back into the cell by active transport. This can also be seen with pathogenic bacteria inside its host, where iron is bound tightly by haemoglobin, transferrin, lactoferrin and ferritin, and thus low in concentration (10⁻²⁴ mol L⁻¹). Here it secrets siderophores which has a higher affinity (with a formation constant, or ([ML])/([M][L]), of 10⁴⁹)to Fe³⁺ than the host's iron-binding proteins, and so will remove iron and then transported inside the cell. Bacillus anthracis, a Gram-positive bacteria^[3] that causes anthrax, secretes two siderophores: bacillibactin and petrobactin. Escherichia coli secrets many iron-siderophore transports, but produce only one siderophore—enterobactin. The ferric enterobactin receptor FepA recognises the catecholate part of ferric enterobactin (FeEnt), and transports it across the outer membrane from the extracellular space into the periplasm. The binding is thought to be in two phases,^[4] a fast step which recognises FeEnt, and a slower step which may be the first step in translocation—preparing the complex for translocation. Both steps occur independently of the TonB–ExbB–ExbD complex and the proton motive force it provides. In the periplasm, FeEnt is bound by FepB and passed to the integral inner membrane proteins FepG and FepD through active transport, with the energy provided by ATP hydrolysis catalysed by cytoplasmic FepC. In the cytoplasm, the Fes enterobactin esterase hydrolyses and this cleaves enterobactin, releasing Fe³⁺ which will subsequently be reduced by the same protein, Fes, to Fe²⁺.

Possible Mechanisms

The 14P of the TonB box is essential for interaction, as its mutation to isoleucine led to null interaction; from this, it was also suggested that the interaction is conformational, and not sequence-specific. As there are a limited number of the TonB–ExbB–ExbD complex, it has been shown that it interacts more favourably with FepA that has a ligand bound to it. The mechanism of transport has been described as similar to an air lock. When the ligand is bound, it closes the pore at the extracellular side, and thus preventing anything from exiting through the pore. FepA then interacts with TonB, which induces a conformational change to the N-terminal, and so open the pore at the periplasmic side. This would allow FepA to transport FeEnt without allowing ions and small molecules from passing in either direction, which would occur if the system contains only a single plug.

References

1. S, Buchanan; B, Smith; L, Venkatramani; D, Xia; L, Esser; M, Palnitkar; R, Chakraborty; D, van der Helm; J, Deisenhofer (1999). "Crystal structure of the outer membrane active transporter FepA from Escherichia coli". Nature Structural Biology 6 (1): 56–63. doi:10.1038/4931. PMID 9886293.
2. Raymond, K; Dertz, E; Kim, S (2003). "Enterobactin: An archetype for microbial iron transport". PNAS 100 (7): 3584–3588. doi:10.1073/pnas.0630018100. PMC 152965. PMID 12655062.
3. Spencer, RC (2003). "Bacillus anthracis". Journal of Clinical Pathology 56 (3): 182–187. doi:10.1136/jcp.56.3.182. PMC 1769905. PMID 12610093.
4. Payne, M; Igo, J; Cao, Z; Foster, S; Newton, S; Klebba, P (1997). "Biphasic Binding Kinetics Between FepA and its Ligands". The Journal of Biological Chemistry 272 (35): 21950–21955. doi:10.1074/jbc.272.35.21950. PMID 9268330.