FhuA
Created by Matthew Ha
FhuA (1by5) is an important siderophore transport receptor found in Escherichia coli that transports ferrichrome, an iron-cyclic hexapeptide complex, across the outer membrane of pathogenic bacteria. The iron found in ferrichrome is essential for a number of metabolic processes such as electron transport, DNA synthesis, and many different redox reactions. Iron deprivation impedes growth and will cause eventual cell death. (9) Because Escherichia coli and other pathogenic bacteria need the iron from ferrichrome and other iron complexes in order to grow, they must have successfully evolved pathways that compete for the iron in host tissues and fluids (1). During an infection in a mammalian body, iron levels are reduced in eukaryotic extracellular and intracellular compartments largely due to bacterial absorption. Utilizing two strategies to compete with eukaryotic environments for iron uptake, bacteria will either scavenge iron from eukaryotic host proteins via transferrin or acquire iron from the environment via siderophores (2).
A siderophore is a small, high-affinity iron chelating (multiple coordination bonds) compound that preferentially complexes with Fe3+ due to its six coordinating bonds. The FhuA protein is an important part of the iron-siderophore transport pathway. The ability of bacteria to acquire and utilize iron is a major determinant in successful virulence (1). The FhuA transport system is the specific target of the antibiotic, albomycin, which is a type of sideromycin that is compose of an iron carrier linked to an antibiotic moiety. (10) The molecular weight of the FhuA protein is 78853.34 Da, and its isoelectric point is 5.18.
In Gram-negative bacteria such as E.coli, both strategies of iron acquisition involve a substrate-specific exoplasmic membrane receptor and an energy-transducing molecule such as the TonB complex found in FhuA that is anchored in the cytoplasmic leaflet. The substrate binds to the extracellular face of the receptor and signals the TonB complex, triggering an energy-driven translocation of the substrate across the plasma membrane. FhuA, the protein of study, falls under this category of TonB-dependent receptors that are vital to the virulence of pathogenic bacteria. The importance of TonB-dependent receptors is evidenced by the presence of multiple genetic copies found in Haemophilus influenza, Heliobacter pylori, and Escherichia coli (2).
To date, at least four TonB-dependent receptors have been solved, three of which are iron-siderophore transporters: ferrichrome transporter FhuA, ferric enterobactin transporter FepA, and ferric dicitrate transporter FecA. The fourth structure is cobalamin transporter BtuB. All of these structures are composed of a conserved N-terminal globular domain and a 22-stranded beta-barrel. The hatch or plug domain is located within the barrel and blocks the large pore of the barrel domain. Using residues from plug loops, from the interior surface of beta-barrel strands and from extracellular barrel loops, the TonB-dependent transporters bind and stabilize the substrate (3).
Ferric hydroxamate uptake protein, FhuA, (from E. Coli) is responsible for the uptake of the siderophore ferrichrome. Ferrichrome is a cyclic hexapeptide composed of three glycine and three modified ornithine residues that bind Fe3+ with hydroxamate groups (6). The energy for the active transport of iron-siderophore complexes across the outer membrane in Gram-negative bacteria is provided by the cytoplasmic membrane proton-motive force. The TonB protein in TonB-dependent receptors provides this proton-motive force. Theoretically, by shuttling between membranes, TonB transduces energy that drives the import of ferrichrome in FhuA (7). The FhuA has only two conformations: a liganded state and an unliganded state (2).
The solved structure of FhuA is a monomer with two domains: a beta-barrel and a plug domain. The beta-barrel acts as a container for the ferrichrome complex and the plug blocks the channel. The FhuA protein is 714 residues in length. The beta-barrel domain covers residues 160-714 and the plug domain covers residues 1-159. Two aromatic residues characterize the internal face of the protein that is exposed to the hydrophobic membrane core. The hydrophobic residues located on the internal face of the beta-barrel and in the binding pocket face of the plug domain are likely responsible for attracting the siderophores from the extracellular medium into the transporter. The beta strands range from 7 to 28 residues in length and the loops range from 3 to 31 residues in length. The loops from the beta-barrel are similar to those found in porins but are substantially longer with much more significant extension into the extracellular area. The “gating loop” bends into and constricts the channel lumen which regulates channel transportation of ferrichrome. The globular plug domain blocks the barrel, and the two domains are connected by a hinge region. The plug is made of five alpha helices and six beta strands, and the folding of the plug is unique to the FhuA protein. The plug domain has more than 60 hydrogen bonds and 9 salt bridge interactions with the barrel lining. (2)
Ferrichrome binds at the apical interface of the plug domain and loops L3 and L11 of the beta-barrel. The aromatic side chains in the binding pocket form strong hydrogen bonds to the substrate, and the siderophore is placed with its complexed iron-side inside the pocket, while its tail remains free in the extracellular area. The siderophore’s conformation changes very little if at all when inside the binding pocket as opposed to when it is free. Ferrichrome is further stabilized in the binding pocket by hydrogen bonding with FhuA plug residues R81, Y116, and G99 and with barrel residues Y244 and Y315. These interactions explain the strong binding of ferrichrome to FhuA (4).
During signal transduction, the pattern of noncovalent bonds within the plug and barrel are altered causing the hinge region and its corresponding segment to swing to the opposite side of the barrel wall. The swing of the hinge region requires two turns of the plug region and a shift of the plug towards the ligand of about 1-2 A (2).
The liganded and unliganded (superimposed) conformations of the protein showed only a difference in the location of the N-terminal TonB box, which shifted significantly with the binding of ferrichrome. Current studies have been unable to determine the exact mechanism for the translocation of ferrichrome across the membrane. The gating loop, L4, is likely to play a significant role in translocation. Deletion of L4 creates a permanently open, TonB-independent channel which means L4 plays a role in plug insertion as well as ferrichrome translocation. The interaction of the L4 gating loop with three other loops, L3, L5, and L11, indicates that they are all involved likely involved in the opening and closing of the channel (2, 5).
Because it is located on the outer membrane, it is also the site of attack of several bacteriophages, bacterial toxins, and antibiotics such as albomycin. (2) Albomycin specifically utilizes the ferric hydroxamate transport system to cross the outer membrane. By covalently linking to iron-chelating siderophores, albomycin is able to cross through the FhuA transporter. Consisting of a hydroxamate-type iron-chelating siderophore, albomycin has an identical coordination geometry as the ferrichrome complex has surrounding the ferric iron atom. The siderophore consists of three parts: an iron-chelating siderophore, a peptide linker, and an antibiotic group. In order to exhibit microbial activity, albomycin has to be cleaved by a peptidase within the cell. Bacterial cells with mutant peptidase activity were albomycin-resistant. The mechanism of albomycin’s antimicrobial activity is not completely understood. (8)
Ferric hydroxamate uptake protein (1by5) has approximately 24% sequence identity and 64% sequence similarity to ferric citrate transporter (1po3). The results of DALI (Z=38.8, rmsd =2.8) and protein blast (E =7e-6) show that FhuA has primary and tertiary similarities to FecA (12). The beta barrel residues 417-714 in FhuA and 440-751 in FecA had approximately 64% sequence conservation, in both siderophore transporters. The tertiary structures of the FhuA and FecA protein are very similar but their functional mechanisms differ. Both proteins have a beta barrel structure made of 22 antiparallel beta strands which is blocked by an N-terminal globular plug domain. There is significantly less sequence conservation, however, in the plug domain and first half of the beta barrel domain of both proteins.
The FecA protein differs in its transport mechanism’s use of gating loops L7 and L8, which close the entrance to the ferric citrate binding site upon binding of the substrate. The gating loops in FhuA did not move significantly during the binding of ferrichrome. Loops 7 (FhuA 14, FecA 18 residues) and 8 (FhuA 7, FecA 20 residues) in both proteins differ in size, giving strong evidence that these loops do not have the same function in both transporters. Loop 7 and 8 deletion mutants completely stop induction and transport activity in the FecA, whereas FhuA was able to function normally without either loop. Interestingly, the FhuA critical loops 3 and 11, however, share significant functional implications in both proteins. Deletion mutants of loops 3 and 11 ended all activities in both proteins. Critical glutamic acid residues are conserved in both proteins in the beta barrel region. E587 in FecA and E522 and E571 in FhuA cannot be replaced with R residues without losing activity in both proteins. These residues likely represent the polar interaction sites between the globular plug domain and the beta-barrel. (11)