Receptor ferrichrome

Mainly the outer membrane ferrichrome receptor and transporter FhuA will be discussed because most structural and functional studies have been performed with this protein. In fact, FhuA was the first outer membrane protein identified (called TonA), with known functions as a phage and colicin receptor, that are related to iron transport (for a historical account, see Braun and Hantke 1977). 

The degeneracy of the non-chiral complexes can be removed by incorporating chiral centers, usually as resolved amino acids, into the arms at close vicinity to the hydroxamate iron binding sites. Thus, only one of the energetically non-equivalent diastereomers predominates, leading to pure enantiomeric iron(III) complexes with defined hehcity that allows assessing stereospecific recognition by the ferrichrome receptor. 

Since modifications at the hydroxamate termini or the projecting side groups on the linking arms were not tolerated by the ferrichrome receptor, most of the modifications were carried out at the epical template site (Figure 6). 

Competition at the Outer Membrane. Ferrichrome Receptor. Escherichia coli. It was easily demonstrated that tonA and tonB mutants were also resistant to albomycin (I, 35). The first experiments to test competition were performed by simply adding ferrichrome to wild type cells plus phage in a plate assay. Later an adsorption assay was used to... 

Figure 10. Diagrammatic sketch of the competition between phage colicin and siderophore for the outer membrane receptor in the enteric bacteria. (A) Escherichia coli K-12. (1) Ferrichrome receptor phage = TI, T5, 80 colicin = M siderophore = ferrichrome. (2) Ferric en-terobactin receptor phage = colicin = B siderophore = ferric entero-bactin. (B) Salmonella typhimurium LT-2. (1) Ferrichrome receptor ...

The maltose molecule is too small to be fitted conveniently into the molecular barrier hypothesis, but the receptor for this substance functions well with maltodextrins which do have higher molecular weights. The receptor is induced with maltose, a substrate which is doubtless commonly encountered in the diet of enteric bacteria. Biologically the receptor serves for transport of maltose and for chemotaxis to this substrate (72). The data presented in Table IV, especially that pertaining to the ferrichrome receptor, demonstrate convincingly that phage receptors were designed for nutritious substances. 

The FhuA receptor of E. coli transports the hydroxamate-type siderophore ferrichrome (see Figure 9), the structural similar antibiotic albomycin and the antibiotic rifamycin CGP 4832. Likewise, FepA is the receptor for the catechol-type siderophore enterobactin. As monomeric proteins, both receptors consist of a hollow, elliptical-shaped, channel-like 22-stranded, antiparallel (3-barrel, which is formed by the large C-terminal domain. A number of strands extend far beyond the lipid bilayer into the extracellular space. The strands are connected sequentially using short turns on the periplasmic side, and long loops on the extracellular side of the barrel. 

Figure 8 (Plate 7). Structure of the Escherichia coli FhuA protein serving as receptor for ferrichrome and the antibiotic albomycin. (a) side view (b) side aspect with partly removed barrel to allow the view on the cork domain (c) top view. A single lipo-polysaccharide molecule is tightly associated with the transmembrane region of FhuA (reproduced by permission of W. Welte and A. Brosig)...

As mentioned above, transport of siderophores across the cytoplasmic membrane is less specific than the translocation through the outer membrane. In E. coli three different outer membrane proteins (among them FepA the receptor for enterobactin produced by most E. coli strains) recognise siderophores of the catechol type (enterobactin and structurally related compounds), while only one ABC system is needed for the passage into the cytosol. Likewise, OM receptors FhuA, FhuE, and Iut are needed to transport a number of different ferric hydroxamates, whereas the FhuBCD proteins accept a variety of hydroxamate type ligands such as albomycin, ferrichrome, coprogen, aerobactin, shizokinen, rhodotorulic acid, and ferrioxamine B [165,171], For the vast majority of systems, the substrate specificity has not been elucidated, but it can be assumed that many siderophore ABC permeases might be able to transport several different but structurally related substrates. 

Locher, K. P., Rees, B., Koebnik, R., Mitschler, A., Moulinier, L., Rosenbusch, J. P. and Moras, D. (1998). Transmembrane signaling across the ligand-gated FhuA receptor crystal structures of free and ferrichrome-bound states reveal allosteric changes, Cell, 95, 771-778. 

Relationships between the structure of the siderophores and the iron transport were investigated for the fungus Neurospora crassa (160, 160a). Apparently two different receptors exist for ferrichromes and for coprogenes. For the recognition and the binding to the cell surface the iron configuration and the nature of the acyl chains is of importance. However, the transport system seems to be the same for both siderophore types dependent on the peptide part of the molecules. 

Since siderophore receptors, being membrane-bound, are hard to crystallize, there are to date few structures in the Protein Data Bank (PDB). One such structure is the E. call FhuA entries IbyS, with and without ferrichrome, Ifcp and 2fcp , respectively. ... 

The FhuA receptor, composed of a -barrel and iV-terminal globular domains, serves as a cork that plugs the barrel, separating the receptor s interior space into inner and outer cavities. The ferrichrome complex forms hydrogen bonding and van der Waals interactions with the outer cavity. 

The closely related structures show completely different microbial uptake characteristics. The 3D structures described above show distinct different orientation of the backbone amide (tangental in type 1 versus radial in type 2), which can be explained by the interactions that take place between the FhuA receptor and the ferrichrome siderophore -As mentioned, the second coordination sphere of natural ferrichrome in FhuA receptor is very sensitive to the distance and orientation between a proton donor and the proton acceptor, therefore the orientation of the amide groups in the biomimetic siderophore plays a crucial role in receptor recognition. 

Since the 1,2-HOPO chelators form neutral complexes while the catecholates form charged complexes, it is reasonable to assume that charged species are essential for the enterobactin receptor recognition. The lack of recognition by the ferrichrome analog may well be attributed to the bulky substituents on the hydroxamate moiety in agreement with early observations by Emery and Emery and others ... 

Two fluorescent siderophore analogs, one based on ferrichrome 173 and the second on ferrioxamine 188, were used to study iron transport in the fungus Ustilago maydis that has an uptake system for ferrichrome but lacks a defined ferrioxamine receptor. Nevertheless, ferrioxamine can be utilized by the fungus albeit at a slower rate. 

Figure 7. Antagonism between ferrichrome and albomycin for membrane receptor in Salmonella typhimurium and the appearance of mutants resistant to the antibiotic (2). Several of these mutants owe their resistance to a defect in ferrichrome transport. The nature of this effect as competition for a surface site was elucidated by Zimmerman and Knusel (32).

The tonB mutation, which had already been connected to iron transport (see above), also affected the outer membrane. Inspection of the region of the E. coli linkage map analogous to the sid locus in S. typhimurium revealed that tonA, the structural gene for the T5 outer membrane receptor, maps near pan. These considerations raised the possibility that the tonA product could be related to ferrichrome and a sid function in E. coli. The characteristics of the ton mutations known at that time, with the exception of albomycin resistance, are recorded in Table I. 

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