Ligand-induced conformational change in the ferrichrome–iron receptor of Escherichia coli K-12

Ferrichrome–iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of -sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.     

Properties of the FhuA channel in the Escherichia coli outer membrane after deletion of FhuA portions within and outside the predicted gating loop.

Escherichia coli transports Fe3+ as a ferrichrome complex through the outer membrane in an energy-dependent process mediated by the FhuA protein. A FhuA deletion derivative lacking residues 322 to 355 (FhuA delta322-355) forms a permanently open channel through which ferrichrome diffused. This finding led to the concept that the FhuA protein forms a closed channel that is opened by input of energy derived from the electrochemical potential across the cytoplasmic membrane, mediated by the Ton system. In this study, we constructed various FhuA derivatives containing deletions inside and outside the gating loop. FhuA delta322-336 bound ferrichrome and displayed a residual Ton-dependent ferrichrome transport activity. FhuA delta335-355 no longer bound ferrichrome but supported ferrichrome diffusion through the outer membrane in the absence of the Ton system. FhuA delta335-355 rendered cells sensitive to sodium dodecyl sulfate and supported diffusion of maltotetraose and maltopentaose in a lamB mutant lacking the maltodextrin-specific channel in the outer membrane. Cells expressing FhuA delta70-223, which has a large deletion outside the gating loop, were highly sensitive to sodium dodecyl sulfate and grew on maltodextrins but showed only weak ferrichrome uptake, suggesting formation of a nonspecific pore through the outer membrane. FhuA delta457-479 supported Ton-dependent uptake of ferrichrome. None of these FhuA deletion derivatives formed pores in black lipid membranes with a stable single-channel conductance. Rather, the conductance displayed a high degree of current noise, indicating a substantial influence of the deletions on the conformation of the FhuA protein. FhuA also supports infection by the phages T1, T5, and phi80 and renders cells sensitive to albomycin and colicin M. Cells expressing FhuA delta322-336 were sensitive to albomycin and colicin M but were only weakly sensitive to T5 and phi480 and insensitive to T1. Cells expressing FhuA delta335-355 were resistant to all FhuA ligands. These results indicate different structural requirements within the gating loop for the various FhuA ligands. Cells expressing FhuA delta457-479 displayed a strongly reduced sensitivity to all FhuA ligands, while cells expressing FhuA delta70-223 were rather sensitive to all FhuA ligands except albomycin, to which they were nearly resistant. It is concluded that residues 335 to 355 mainly determine the properties of the gate with regard to FhuA permeability and ligand binding.     

Conversion of the FhuA transport protein into a diffusion channel through the outer membrane of Escherichia coli.

The FhuA receptor protein is involved in energy-coupled transport of Fe3+ via ferrichrome through the outer membrane of Escherichia coli. Since no energy source is known in the outer membrane it is assumed that energy is provided through the action of the TonB, ExbB and ExbD proteins, which are anchored to the cytoplasmic membrane. By deleting 34 amino acid residues of a putative cell surface exposed loop, FhuA was converted from a ligand specific transport protein into a TonB independent and nonspecific diffusion channel. The FhuA deletion derivative FhuA delta 322-355 formed stable channels in black lipid membranes, in contrast to wild-type FhuA which did not increase membrane conductance. The single-channel conductance of the FhuA mutant channels was at least three times larger than that of the general diffusion porins of E. coli outer membrane. It is proposed that the basic structure of FhuA in the outer membrane is a channel formed by beta-barrels. Since the loop extending from residue 316 to 356 is part of the active site of FhuA, it probably controls the permeability of the channel. The transport-active conformation of FhuA is mediated by a TonB-induced conformational change in response to the energized cytoplasmic membrane. The ferrichrome transport rate into cells expressing FhuA delta 322-355 increased linearly with increasing substrate concentration (from 0.5 to 20 microM), in contrast to FhuA wild-type cells, which displayed saturation at 5 microM. This implies that in wild-type cells ferrichrome transport through the outer membrane is the rate-limiting step and that TonB, ExbB and ExbD are only required for outer membrane transport.     

Specific In Vivo Labeling of Cell Surface-Exposed Protein Loops: Reactive Cysteines in the Predicted Gating Loop Mark a Ferrichrome Binding Site and a Ligand-Induced Conformational Change of the Escherichia coli FhuA Protein

The FhuA protein of Escherichia coli K-12 transports ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5, 80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It is thought that the proteins TonB, ExbB, and ExbD function as a coupling device between the cytoplasmic membrane and the outer membrane. Excision of 34 residues from FhuA, tentatively designated the gating loop, converts FhuA into a permanently open channel. FhuA contains two disulfide bridges, one in the gating loop and one close to the C-terminal end. Reduction of the disulfide bridges results in a low in vivo reaction of the cysteines in the gating loop and no reaction of the C-terminal cysteines with biotin-maleimide, as determined by streptavidin-β-galactosidase bound to biotin. In this study we show that a cysteine residue introduced into the gating loop by replacement of Asp-336 displayed a rather high reactivity and was used to monitor structural changes in FhuA upon binding of ferrichrome. Flow cytometric analysis revealed fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by ferrichrome, but ferrichrome quenching was negligible. It is concluded that binding of ferrichrome causes a conformational change of the gating loop and that Cys-347 is part of or close to the ferrichrome binding site. Fluorescence quenching was independent of the TonB activity. The newly introduced cysteines and the replacement of the existing cysteines by serine did not alter sensitivity of cells to the FhuA ligands tested (T5, 80, T1, colicin M, and albomycin) and fully supported growth on ferrichrome as the sole iron source. Since cells of E. coli K-12 display no reactivity to thiol reagents, newly introduced cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.     

FhuA, a transporter of the Escherichia coli outer membrane, is converted into a channel upon binding of bacteriophage T5.

The Escherichia coli outer membrane protein FhuA catalyzes the transport of Fe3+(-)ferrichrome and is the receptor of phage T5 and phi 80. The purified protein inserted into planar lipid bilayers showed no channel activity. Binding of phage T5 and FhuA resulted in the appearance of high conductance ion channels. The electrophysiological characteristics of the channels (conductance, kinetic behavior, substates, ion selectivity including the effect of ferrichrome) showed similarities with those of the channel formed by a FhuA derivative from which the 'gating loop' (delta 322-355) had been removed. binding of phage T5 to FhuA in E.coli cells conferred SDS sensitivity to the bacteria, suggesting that such channels also exist in vivo. These data suggest that binding of T5 to loop 322-355 of FhuA, which constitutes the T5 binding site, unmasks an inner channel in FhuA. Both T5 and ferrichrome bind to the closed state of the channel but only T5 can trigger its opening.     

Active transport of an antibiotic rifamycin derivative by the outer-membrane protein FhuA

BACKGROUND: FhuA, an integral membrane protein of Escherichia coli, actively transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane. The transport is coupled to the proton motive force, which energizes FhuA through the inner-membrane protein TonB. FhuA also transports the semisynthetic rifamycin derivative CGP 4832, although the chemical structure of this antibiotic differs markedly from that of ferric hydroxamates. RESULTS: X-ray crystallography revealed that rifamycin CGP 4832 occupies the same ligand binding site as ferrichrome and albomycin, thus demonstrating a surprising lack of selectivity. However, the binding of rifamycin CGP 4832 is deviant from the complexes of FhuA with hydroxamate-type ligands in that it does not result in the unwinding of the switch helix but only in its destabilization, as reflected by increased B factors. Unwinding of the switch helix is proposed to be required for efficient binding of TonB to FhuA and for coupling the proton motive force of the cytoplasmic membrane with energy-dependent ligand transport. The transport data from cells expressing mutant FhuA proteins indicated conserved structural and mechanistic requirements for the transport of both types of compounds. CONCLUSIONS: We conclude that the binding of rifamycin CGP 4832 destabilizes the switch helix and promotes the formation of a transport-competent FhuA-TonB complex, albeit with lower efficiency than ferrichrome. Active transport of this rifamycin derivative explains the 200-fold increase in potency as compared to rifamycin, which is not a FhuA-specific ligand and permeates across the cell envelope by passive diffusion only.     

Conversion of the coprogen transport protein FhuE and the ferrioxamine B transport protein FoxA into ferrichrome transport proteins

The FhuA protein of Escherichia coli K-12 transports ferrichrome and the structurally related antibiotic albomycin across the outer membrane and serves as a receptor for the phages T1, T5, and φ80 and for colicin M. In this paper, we show that chimeric proteins consisting of the central part of FhuA and the N- and C-terminal parts of FhuE (coprogen receptor) or the N- and/or C-terminal parts of FoxA (ferrioxamine B receptor), function as ferrichrome transport proteins. Although the hybrid proteins contained the previously identified gating loop of FhuA, which is the principal binding site of the phages T5, T1, and φ80, only the hybrid protein consisting of the N-terminal third of FoxA and the C-terminal two thirds of FhuA conferred weak phage sensitivity to cells. Apparently, the gating loop is essential, but not sufficient for wild-type levels of ferrichrome transport and for phage sensitivity. The properties of FhuA-FoxA hybrids suggest different regions of the two receptors for ferric siderophore uptake.     

The β-barrel domain of FhuAΔ5-160 is sufficient for TonB-dependent FhuA activities of Escherichia coli

FhuA in the outer membrane of Escherichia coli serves as a transporter for ferrichrome, the antibiotics albomycin and rifamycin CGP4832, colicin M, and as receptor for phages T1, T5 and phi80. The previously determined crystal structure reveals that residues 160-714 of the mature protein form a beta-barrel that is closed from the periplasmic side by the globular N-proximal fragment, residues 1-159, designated the cork. In this study, deletion of the cork resulted in a stable protein, FhuADelta5-160, that was incorporated in the outer membrane. Cells that synthesized FhuADelta5-160 displayed a higher sensitivity to large antibiotics such as erythromycin, rifamycin, bacitracin and vancomycin, and grew on maltotetraose and maltopentaose in the absence of LamB. Higher concentrations of ferrichrome supported growth of a tonB mutant that synthesized FhuADelta5-160. These results demonstrate non-specific diffusion of compounds across the outer membrane of cells that synthesize FhuADelta5-160. However, growth of a FhuADelta5-160 tonB wild-type strain occurred at low ferrichrome concentrations, and ferrichrome was transported at about 45% of the FhuA wild-type rate despite the lack of ferrichrome binding sites provided by the cork. FhuADelta5-160 conferred sensitivity to the phages and colicin M at levels similar to that of wild-type FhuA, and to albomycin and rifamycin CGP 4832. The activity of FhuADelta5-160 depended on TonB, although the mutant lacks the TonB box (residues 7-11) previously implicated in the interaction of FhuA with TonB. CCCP inhibited tonB-dependent transport of ferrichrome through FhuADelta5-160. FhuADelta5-160 still functions as a specific transporter, and sites in addition to the TonB box are involved in the TonB-mediated response of FhuA to the proton gradient of the cytoplasmic membrane. It is proposed that TonB interacts with the TonB box of FhuA and with the beta-barrel to release ferrichrome from the FhuA binding sites and to open the channel in FhuA. For transport of ferrichrome through the open channel of FhuADelta5-160, interaction of TonB with the beta-barrel is sufficient to release ferrichrome from the residual binding sites at the beta-barrel and to induce the active conformation of the L4 loop at the cell surface for infection by the TonB-dependent phages T1 and phi80.     

Iron (III) hydroxamate transport into Escherichia coli. Substrate binding to the periplasmic FhuD protein

Due to its extreme insolubility, Fe3+ is not transported as a monoatomic ion. In microbes, iron is bound to low molecular weight carriers, designated siderophores. For uptake into cells of Escherichia coli Fe3+ siderophores have to be translocated across two membranes. Transport across the outer membrane is receptor-dependent and energy-coupled; transport across the cytoplasmic membrane seems to follow a periplasmic binding protein-dependent transport mechanism. In support of this notion we demonstrate specific binding of the Fe3+ hydroxamate compounds ferrichrome, aerobactin, and coprogen, which are transported via the Fhu system, to the periplasmic FhuD protein, and no binding of the transport inactive ferrichrome A, ferric citrate, and iron sulfate. About 10(4) ferrichrome molecules were bound to the FhuD protein of cells which overproduced plasmid-encoded FhuD. Binding depended on transport across the outer membrane mediated by the FhuA receptor and the TonB protein. Binding to FhuD was supported by the exclusive resistance of FhuD to proteinase K in the presence of the transport active hydroxamates. The overproduced precursor form of the FhuD protein was not protected by the Fe3+ hydroxamates indicating a conformation different to the mature form. The FhuD protein apparently serves as a periplasmic carrier for Fe3+ hydroxamates with widely different structures.     

Enhanced binding of TonB to a ligand-loaded outer membrane receptor: role of the oligomeric state of TonB in formation of a functional FhuA.TonB complex

The ferric hydroxymate uptake (FhuA) receptor from Escherichia coli facilitates transport of siderophores ferricrocin and ferrichrome and siderophore-antibiotic conjugates such as albomycin and rifamycin CGP 4832. FhuA is also the receptor for phages T5, T1, Phi80, UC-1, for colicin M and for the antimicrobial peptide microcin MccJ21. Energy for transport is provided by the cytoplasmic membrane complex TonB.ExbB.ExbD, which uses the proton motive force of the cytoplasmic membrane to transduce energy to the outer membrane. To accomplish energy transfer, TonB contacts outer membrane receptors. However, the stoichiometry of TonB. receptor complexes and their sites of interaction remain uncertain. In this study, analyses of FhuA interactions with two recombinant TonB proteins by analytical ultracentrifugation revealed that TonB forms a 2:1 complex with FhuA. The presence of the FhuA-specific ligand ferricrocin enhanced the amounts of complex but is not essential for its formation. Surface plasmon resonance experiments demonstrated that FhuA.TonB interactions are multiple and have apparent affinities in the nanomolar range. TonB also possesses two distinct binding regions: one in the C terminus of the protein, for which binding to FhuA is ferricrocin-independent, and a higher affinity region outside the C terminus, for which ferricrocin enhances interactions with FhuA. Together these experiments establish that FhuA.TonB interactions are more intricate than originally predicted, that the TonB.FhuA stoichiometry is 2:1, and that ferricrocin modulates binding of FhuA to TonB at regions outside the C-terminal domain of TonB.     

Determination of ferrichrome binding to the FhuA outer membrane transport protein,periplasmic accumulation of ferrichrome,or transport of ferrichrome into cells using a three-layer oil technique

A new method for the determination of ferrichrome binding to the FhuA transporter in the Escherichia coli outer membrane, ferrichrome accumulation in the periplasmic space, and ferrichrome transport into the cytoplasm was developed. Cells were separated from residual, soluble, radiolabeled ferrichrome by centrifugation in a micro-test tube containing three layers of nonmixable solutions of different densities. Cells in the upper aqueous layer passed through the middle silicone oil layer, but did not enter the underlying NaI layer, thereby accumulating on top of the NaI layer; soluble compounds remained in the upper aqueous layer. Cells were then easily recovered by centrifugation, and radioactivity was determined by liquid scintillation counting. Reproducible results for all applications tested were obtained without the need for any washing steps. The method was tested by determination of receptor binding and transport of ferrichrome with various FhuA mutants which, in contrast to their transport activity, showed only a weak binding of ferrichrome to FhuA and compared with the commonly used cellulose nitrate filter method. Similar transport rates were obtained with the two methods, but binding of ferrichrome to the mutated FhuA proteins and accumulation of ferrichrome in the periplasm could be measured only with the new method.     

Mutual inhibition of cobalamin and siderophore uptake systems suggests their competition for TonB function.

Vitamin B12 (CN-Cbl) and iron-siderophore complexes are transported into Escherichia coli in two energy-dependent steps. The first step is mediated by substrate-specific outer membrane transport proteins and the energy-coupling TonB protein complex, and the second step uses separate periplasmic permeases for transport across the cytoplasmic membrane. Genetic and biochemical evidence suggests that the TonB-dependent outer membrane transporters contact TonB directly, and thus they might compete for limiting amounts of functional TonB. The transport of iron-siderophore complexes, such as ferrichrome, causes a partial decrease in the rate of CN-Cbl transport. Although CN-Cbl uptake does not inhibit ferrichrome uptake in wild-type cells, in which the amount of the outer membrane ferrichrome transporter FhuA far exceeds that of the cobalamin transporter BtuB, CN-Cbl does inhibit ferrichrome uptake when BtuB is overexpressed from a multicopy plasmid. This inhibition by CN-Cbl is increased when the expression of FhuA and TonB is repressed by growth with excess iron and is eliminated when BtuB synthesis is repressed by CN-Cbl. The mutual inhibition of CN-Cbl and ferrichrome uptake is overcome by increased expression of TonB. Additional evidence for interaction of the Cbl and iron transport systems is provided by the strong stimulation of the BtuB- and TonB-dependent transport of CN-Cbl into a nonexchangeable, presumably cytoplasmic pool by preincubation of cells with the iron(II) chelator 2,2'-dipyridyl. Other metal ion chelators inhibited CN-Cbl uptake across the outer membrane. Although the effects of chelators are multiple and complex, they indicate competition or interaction among TonB-dependent transport systems.     

Diffusion through channel derivatives of the Escherichia coli FhuA transport protein.

FhuA is a multifunctional protein in the outer membrane of Escherichia coli that actively transports [Fe3+]ferrichrome, the antibiotics albomycin and rifamycin CGP 4832, and mediates sensitivity of cells to the unrelated phages T5, T1, phi80 and UC-1, and to colicin M and microcin J25. The energy source of active transport is the proton motive force of the cytoplasmic membrane that is required for all FhuA functions except for infection by phage T5. The FhuA crystal structure reveals 22 antiparallel transmembrane beta-strands that form a beta-barrel which is closed by a globular N-terminal domain. FhuA still displays active transport and sensitivity to all ligands except microcin J25 when the globular domain (residues 5-160) is excised and supports weakly unspecific diffusion of substrates across the outer membrane. Here it is shown that isolated FhuADelta5-160 supported diffusion of ions through artificial planar lipid bilayer membranes but did not form stable channels. The double mutant FhuADelta5-160 Delta322-336 lacking in addition to the globular domain most of the large surface loop 4 which partially constricts the channel entrance, displayed an increased single-channel conductance but formed no stable channels. It transported in vivo[Fe3+]ferrichrome with 45% of the rate of wild-type FhuA and did not increase sensitivity of cells to antibiotics. In contrast, a second FhuA double mutant derivative which in addition to the globular domain contained a deletion of residues 335-355 comprising one-third of surface loop 4 and half of the transmembrane beta-strand 8 formed stable channels in lipid bilayers with a large single-channel conductance of 2.5 nS in 1 m KCl. Cells that synthesized FhuADelta5-160 Delta335-355 showed an increased sensitivity to antibiotics and supported diffusion of maltodextrins, SDS and ferrichrome across the outer membrane. FhuADelta5-160 Delta335-355 showed no FhuA specific functions such as active transport of [Fe3+]ferrichrome or sensitivity to the other FhuA ligands. It is concluded that FhuADelta5-160 Delta335-355 assumes a conformation that is incompatible with any of the FhuA functions.     

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies.

Ferrichrome-iron transport in Escherichia coli is initiated by the outer membrane receptor FhuA. Thirty-five anti-FhuA monoclonal antibodies (MAbs) were isolated to examine the surface accessibility of FhuA sequences and their contribution to ligand binding. The determinants of 32 of the MAbs were mapped to eight distinct regions in the primary sequence of FhuA by immunoblotting against (i) five internal deletion FhuA proteins and (ii) four FhuA peptides generated by cyanogen bromide cleavage. Two groups of MAbs bound to FhuA in outer membrane vesicles but not to intact cells, indicating that their determinants, located between residues 1 and 20 and 21 and 59, are exposed to the periplasm. One of the 28 strongly immunoblot-reactive MAbs bound to FhuA on intact cells in flow cytometry, indicating that its determinant, located between amino acids 321 and 381, is cell surface exposed. This MAb and four others which in flow cytometry bound to cells expressing FhuA were tested for the ability to block ligand binding. While no MAb inhibited growth promotion by ferrichrome or cell killing by microcin 25, some prevented killing by colicin M and were partially able to inhibit the inactivation of T5 phage. These data provide evidence for spatially distinct ligand binding sites on FhuA. The lack of surface reactivity of most of the immunoblot-reactive MAbs suggests that the majority of FhuA sequences which lie external to the outer membrane may adopt a tightly ordered organization with little accessible linear sequence.     

Inactivation of FhuA at the cell surface of Escherichia coli K-12 by a phage T5 lipoprotein at the periplasmic face of the outer membrane.

Inactivation of phage T5 by lysed cells after phage multiplication is prevented by a phage-encoded lipoprotein (Llp) that inactivates the FhuA outer membrane receptor protein (K. Decker, V. Krauel, A. Meesmann, and K. Heller, Mol. Microbiol. 12:321-332, 1994). Using FhuA derivatives carrying insertions of 4 and 16 amino acid residues and point mutations, we determined whether FhuA inactivation is caused by binding of Llp to FhuA and which regions of FhuA are important for inactivation by Llp. Cells expressing Llp were resistant not only to phage T5 but to all FhuA ligands tested, such as phage phi 80, colicin M, and albomycin, and they were strongly reduced in the uptake of ferrichrome. Most of the FhuA derivatives which were not affected by Llp were, according to a previously published FhuA transmembrane topology model, located in periplasmic turns and in the TonB box close to the periplasm. Since the ligands bind to the cell surface, interaction of FhuA with Llp in the periplasm may induce a FhuA conformation which impairs binding of the ligands. This conclusion was supported by the increase rather than decrease of colicin M sensitivity of two mutants in the presence of Llp. The only Llp-resistant FhuA derivatives with mutations at the cell surface contained insertions of 16 residues in the loop that determines the permeability of the FhuA channel and serves as the principal binding site for all FhuA ligands. This region may be inactivated by steric hindrance in that a portion of Llp penetrates into the channel. Outer membranes prepared with 0.25% Triton X-100 from cells expressing Llp contained inactivated FhuA, suggesting Llp to be an outer membrane protein whose interaction with FhuA was not abolished by Triton X-100. Llp solubilized in 1.1% octylglucoside prevented T5 inactivation by FhuA dissolved in octylglucoside.     

Membrane proteins: A tale of barrels and corks

Crystal structures have been solved for two bacterial outer membrane proteins, FhuA and FepA, which mediate active transport of chelated iron. Analysis of ligand-induced changes in the structure of FhuA has provided our first structural insights into an active transport mechanism for a complex solute.     

Mutant analysis of the Escherichia coli FhuA protein reveals sites of FhuA activity

The FhuA outer membrane protein of Escherichia coli actively transports ferrichrome, albomycin, and rifamycin CGP 4832, and confers sensitivity to microcin J25, colicin M, and the phages T1, T5, and phi80. Guided by the FhuA crystal structure and derived predictions on how FhuA might function, mutants were isolated in the cork domain (residues 1 to 160) and in the beta-barrel domain (residues 161 to 714). Deletion of the TonB box (residues 7 to 11) completely inactivated all TonB-dependent functions of FhuA. Fixation of the cork to turn 7 of the barrel through a disulfide bridge between introduced C27 and C533 residues abolished ferrichrome transport, which was restored by reduction of the disulfide bond. Deletion of residues 24 to 31, including the switch helix (residues 24 to 29), which upon binding of ferrichrome to FhuA undergoes a large structural transition (17 A) and exposes the N terminus of FhuA (TonB box) to the periplasm, reduced FhuA transport activity (79% of the wild-type activity) but conferred full sensitivity to colicin M and the phages. Duplication of residues 23 to 30 or deletion of residues 13 to 20 resulted in FhuA derivatives with properties similar to those of FhuA with a deletion of residues 24 to 31. However, a frameshift mutation that changed QSEA at positions 18 to 21 to KKAP abolished almost completely most of FhuA's activities. The conserved residues R93 and R133 among energy-coupled outer membrane transporters are thought to fix the cork to the beta-barrel by forming salt bridges to the conserved residues E522 and E571 of the beta-barrel. Proteins with the E522R and E571R mutations were inactive, but inactivity was not caused by repulsion of R93 by R522 and R571 and of R133 by R571. Point mutations in the cork at sites that move or do not move upon the binding of ferrichrome had no effect or conferred only slightly reduced activities. It is concluded that the TonB box is essential for FhuA activity. The TonB box region has to be flexible, but its distance from the cork domain can greatly vary. The removal of salt bridges between the cork and the barrel affects the structure but not the function of FhuA.     

Internal deletions in the FhuA receptor of Escherichia coli K-12 define domains of ligand interactions.

The ferrichrome-iron receptor encoded by the fhuA gene of Escherichia coli K-12 is a multifunctional outer membrane receptor required for the binding and uptake of ferrichrome and bacteriophages T5, T1, phi 80, and UC-1 as well as colicin M. To identify domains of the protein which are important for FhuA activities, a library of 31 overlapping deletion mutants in the fhuA gene was generated. Export of FhuA deletion proteins to the outer membrane and receptor functions of the deletion proteins were analyzed. All but three of the deletion mutant FhuA proteins cofractionated with the outer membrane; no FhuA proteins were detected in outer membrane preparations or in cell extracts when the deletions spanned amino acids 418 to 440. Most deletion proteins were susceptible to cleavage by endogenous proteolytic activity; some degradation products were detected on Coomassie blue-stained gels and on Western blots (immunoblots). Receptor functions were measured with the mutated genes present on multicopy plasmids. Two deletion mutants, FhuA delta 060-069 and FhuA delta 129-168, conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating of bacteriophages as that of wild-type FhuA; killing by colicin M was also unaffected. For FhuA delta 021-128 and FhuA delta 406-417, reduced sensitivity to colicin M was detected; wild-type phenotypes were observed for all other FhuA functions. Deletions from amino acids 169 to 195 slightly reduced sensitivities to bacteriophages and to colicin M; ferrichrome growth promotion was unaffected. When deletions extended into the region of amino acids 196 to 405, all FhuA functions were either reduced or abolished. The results indicate that selected regions of the FhuA protein have receptor activities and demonstrate the presence of both shared and unique ligand-responsive domains.