TonB Box Research Articles

NagA-dependent uptake of N-acetyl-glucosamine and N-acetyl-chitin oligosaccharides across the outer membrane of Caulobacter crescentus.

Among the 67 predicted TonB-dependent outer membrane transporters of Caulobacter crescentus, NagA was found to be essential for growth on N-acetyl-beta-D-glucosamine (GlcNAc) and larger chitin oligosaccharides. NagA (93 kDa) has a predicted typical domain structure of an outer membrane transport protein: a signal sequence, the TonB box EQVVIT, a hatch domain of 147 residues, and a beta-barrel composed of 22 antiparallel beta-strands linked by large surface loops and very short periplasmic turns. Mutations in tonB1 and exbBD, known to be required for maltose transport via MalA in C. crescentus, and in two additional predicted tonB genes (open reading frames cc2327 and cc3508) did not affect NagA-mediated GlcNAc uptake. nagA is located in a gene cluster that encodes a predicted PTS sugar transport system and two enzymes that convert GlcNAc-6-P to fructose-6-P. Since a nagA insertion mutant did not grow on and transport GlcNAc, diffusion of GlcNAc through unspecific porins in the outer membrane is excluded. Uptake of GlcNAc into tonB and exbBD mutants and reduction but not abolishment of GlcNAc transport by agents which dissipate the electrochemical potential of the cytoplasmic membrane (0.1 mM carbonyl cyanide 3-chlorophenylhydrazone and 1 mM 2,4-dinitrophenol) suggest diffusion of GlcNAc through a permanently open pore of NagA. Growth on (GlcNAc)(3) and (GlcNAc)(5) requires ExbB and ExbD, indicating energy-coupled transport by NagA. We propose that NagA forms a small pore through which GlcNAc specifically diffuses into the periplasm and functions as an energy-coupled transporter for the larger chitin oligosaccharides.

TonB-dependent maltose transport by Caulobacter crescentus

We have shown previously that Caulobacter crescentus grows on maltodextrins which are actively transported across the outer membrane by the MalA protein. Evidence for energy-coupled transport was obtained by deletion of the exbB exbD genes which abolished transport. However, removal of the TonB protein, which together with the ExbB ExbD proteins is predicted to form an energy-coupling device between the cytoplasmic membrane and the outer membrane, left transport unaffected. Here we identify an additional tonB gene encoded by the cc2334a ORF, which when deleted abolished maltose transport. MalA contains a TonB box that reads EEVVIT and is predicted to interact with TonB. Replacement of valine number 15 in the TonB box by proline abolished maltose transport. Maltose was transported across the cytoplasmic membrane by the MalY protein (CC2283). Maltose transport was induced by maltose and repressed by the MalI protein (CC2284). In addition to MalA, MalY and MalI, the mal locus encodes two predicted cytoplasmic alpha-amylases (CC2285 and CC2286) and a periplasmic glucoamylase (CC2282). The TonB dependence together with the previously described ExbB ExbD dependence demonstrates energy-coupled maltose transport across the outer membrane. MalY is involved in maltose transport across the cytoplasmic membrane by a presumably ion-coupled mechanism.

Predicting the Complex Structure and Functional Motions of the Outer Membrane Transporter and Signal Transducer FecA

Escherichia coli requires an efficient transport and signaling system to successfully sequester iron from its environment. FecA, a TonB-dependent protein, serves a critical role in this process: first, it binds and transports iron in the form of ferric citrate, and second, it initiates a signaling cascade that results in the transcription of several iron transporter genes in interaction with inner membrane proteins. The structure of the plug and barrel domains and the periplasmic N-terminal domain (NTD) are separately available. However, the linker connecting the plug and barrel and the NTD domains is highly mobile, which may prevent the determination of the FecA structure as a whole assembly. Here, we reduce the conformation space of this linker into most probable structural models using the modeling tool CABS, then apply normal-mode analysis to investigate the motions of the whole structure of FecA by using elastic network models. We relate the FecA domain motions to the outer-inner membrane communication, which initiates transcription. We observe that the global motions of FecA assign flexibility to the TonB box and the NTD, and control the exposure of the TonB box for binding to the TonB inner membrane protein, suggesting how these motions relate to FecA function. Our simulations suggest the presence of a communication between the loops on both ends of the protein, a signaling mechanism by which a signal could be transmitted by conformational transitions in response to the binding of ferric citrate.

Modulation by Substrates of the Interaction between the HasR Outer Membrane Receptor and Its Specific TonB-like Protein, HasB

TonB is a cytoplasmic membrane protein required for active transport of various essential substrates such as heme and iron siderophores through the outer membrane receptors of Gram-negative bacteria. This protein spans the periplasm, contacts outer membrane transporters by its C-terminal domain, and transduces energy from the protonmotive force to the transporters. The TonB box, a relatively conserved sequence localized on the periplasmic side of the transporters, has been shown to directly contact TonB. While Serratia marcescens TonB functions with various transporters, HasB, a TonB-like protein, is dedicated to the HasR transporter. HasR acquires heme either freely or via an extracellular heme carrier, the hemophore HasA, that binds to HasR and delivers heme to the transporter. Here, we study the interaction of HasR with a HasB C-terminal domain and compare it with that obtained with a TonB C-terminal fragment. Analysis of the thermodynamic parameters reveals that the interaction mode of HasR with HasB differs from that with TonB, the difference explaining the functional specificity of HasB for HasR. We also demonstrate that the presence of the substrate on the extracellular face of the transporter modifies, via enthalpy–entropy compensation, the interaction with HasB on the periplasmic face. The transmitted signal depends on the nature of the substrate. While the presence of heme on the transporter modifies only slightly the nature of interactions involved between HasR and HasB, hemophore binding on the transporter dramatically changes the interactions and seems to locally stabilize some structural motifs. In both cases, the HasR TonB box is the target for those modifications.

Dual Roles of the Central Domain of Colicin D tRNase in TonB-mediated Import and in Immunity

Colicin D import into Escherichia coli requires an interaction via its TonB box with the energy transducer TonB. Colicin D cytotoxicity is inhibited by specific tonB mutations, but it is restored by suppressor mutations in the TonB box. Here we report that there is a second site of interaction between TonB and colicin D, which is dependent upon a 45-amino acid region, within the uncharacterized central domain of colicin D. In addition, the 8th amino acids of colicin D (a glycine) and colicin B (a valine), adjacent to their TonB boxes, are also required for TonB recognition, suggesting that high affinity complex formation involves multiple interactions between these colicins and TonB. The central domain also contributes to the formation of the immunity complex, as well as being essential for uptake and thus killing. Colicin D is normally secreted in association with the immunity protein, and this complex involves the following two interactions: a major interaction with the C-terminal tRNase domain and a second interaction involving the central domain of colicin D and, most probably, the alpha4 helix of ImmD, which is on the opposite side of ImmD compared with the major interface. In contrast, formation of the immunity complex with the processed cytotoxic domain, the form expected to be found in the cytoplasm after colicin D uptake, requires only the major interaction. Klebicin D has, like colicin D, a ribonuclease activity toward tRNAArg and a central domain, which can form a complex with ImmD but which does not function in TonB-mediated transport.

A β Strand Lock Exchange for Signal Transduction in TonB-Dependent Transducers on the Basis of a Common Structural Motif

Transport of molecules larger than 600 Da across the outer membrane involves TonB-dependent receptors and TonB-ExbB-ExbD of the inner membrane. The transport is energy consuming, and involves direct interactions between a short N-terminal sequence of receptor, called the TonB box, and TonB. We solved the structure of the ferric pyoverdine (Pvd-Fe) outer membrane receptor FpvA from Pseudomonas aeruginosa in its apo form. Structure analyses show that residues of the TonB box are in a beta strand which interacts through a mixed four-stranded beta sheet with the periplasmic signaling domain involved in interactions with an inner membrane sigma regulator. In this conformation, the TonB box cannot form a four-stranded beta sheet with TonB. The FhuA-TonB or BtuB-TonB structures show that the TonB-FpvA interactions require a conformational change which involves a beta strand lock-exchange mechanism. This mechanism is compatible with movements of the periplasmic domain deduced from crystallographic analyses of FpvA, FpvA-Pvd, and FpvA-Pvd-Fe.

Observations on the Calcium Dependence and Reversibility of Cobalamin Transport across the Outer Membrane of Escherichia coli

The calcium dependence of cobalamin (Cbl) binding to the BtuB protein of Escherichia coli and the reversibility of its function in the transport of Cbl across the outer membrane have been examined. The results show that the two calcium-binding sites in BtuB that were identified previously by others are responsible for the calcium dependence of high affinity Cbl binding. The affinity of the pure BtuB protein for Cbl was approximately 1000-fold higher in the presence of saturating levels of calcium than in its absence. The affinities of BtuB for both Cbl and calcium were decreased by insertion of alanine residues at position 51 of the mature protein and were increased by several mutations and deletions in the TonB box. Experiments on the uptake of Cbl into the periplasmic space showed that this process is reversible and that the exit of Cbl back into the medium does not require the protonmotive force. Our interpretation of these results is that the role of the TonB-ExbB-ExbD complex, potentiated by the protonmotive force, is to reduce the affinity of the Cbl-binding site, thus increasing the rate of Cbl release into the periplasmic space. The evidence also indicates that access of the Cbl-binding site of BtuB to the periplasmic space does not require removal of the hatch domain from the barrel.

Studies on colicin B translocation: FepA is gated by TonB

Colicin B is a 55 kDa dumbbell-shaped protein toxin that uses the TonB system (outer membrane transporter, FepA, and three cytoplasmic membrane proteins TonB/ExbB/ExbD) to enter and kill Escherichia coli. FepA is a 22-stranded beta-barrel with its lumen filled by an amino-terminal globular domain containing an N-terminal semiconserved region, known as the TonB box, to which TonB binds. To investigate the mechanism of colicin B translocation across the outer membrane, we engineered cysteine (Cys) substitutions in the globular domain of FepA. Colicin B caused increased exposure to biotin maleimide labelling of all Cys substitutions, but to different degrees, with TonB as well as the FepA TonB box required for all increases. Because of the large increases in exposure for Cys residues from T13 to T51, we conclude that colicin B is translocated through the lumen of FepA, rather than along the lipid-barrel interface or through another protein. Part of the FepA globular domain (residues V91-V142) proved relatively refractory to labelling, indicating either that the relevant Cys residues were sequestered by an unknown protein or that a significant portion of the FepA globular domain remained inside the barrel, requiring concomitant conformational rearrangement of colicin B during its translocation. Unexpectedly, TonB was also required for colicin-induced exposure of the FepA TonB box, suggesting that TonB binds FepA at a different site prior to interaction with the TonB box.

Deletion and Substitution Analysis of theEscherichia coliTonB Q160 Region

The active transport of iron siderophores and vitamin B(12) across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residue-deletion mutant protein (TonBDelta7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonBDelta7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.

Energy-coupled outer membrane transport proteins and regulatory proteins

FhuA and FecA are two examples of energy-coupled outer membrane import proteins of gram-negative bacteria. FhuA transports iron complexed by the siderophore ferrichrome and serves as a receptor for phages, a toxic bacterial peptide, and a toxic protein. FecA transports diferric dicitrate and regulates transcription of an operon encoding five ferric citrate (Fec) transport genes. Properties of FhuA mutants selected according to the FhuA crystal structure are described. FhuA mutants in the TonB box, the hatch, and the beta-barrel are rather robust. TonB box mutants in FhuA FecA, FepA, Cir, and BtuB are compared; some mutations are suppressed by mutations in TonB. Mutant studies have not revealed a ferrichrome diffusion pathway, and tolerance to mutations in the region linking the TonB box to the hatch does not disclose a mechanism for how energy transfer from the cytoplasmic membrane to FhuA changes the conformation of FhuA such that bound substrates are released, the pore is opened, and substrates enter the periplasm, or how surface loops change their conformation such that TonB-dependent phages bind irreversibly and release their DNA into the cells. The FhuA and FecA crystal structures do not disclose the mechanism of these proteins, but they provide important information for specific functional studies. FecA is also a regulatory protein that transduces a signal from the cell surface into the cytoplasm. The interacting subdomains of the proteins in the FecA --> FecR --> FecI --> RNA polymerase signal transduction pathway resulting in fecABCDE transcription have been determined. Energy-coupled transporters transport not only iron and vitamin B12, but also other substrates of very low abundance such as sugars across the outer membrane; transcription regulation of the transport genes may occur similarly to that of the Fec transport genes.

From the Periplasmic Signaling Domain to the Extracellular Face of an Outer Membrane Signal Transducer of Pseudomonas aeruginosa: Crystal Structure of the Ferric Pyoverdine Outer Membrane Receptor

The pyoverdine outer membrane receptor, FpvA, from Pseudomonas aeruginosa translocates ferric pyoverdine across the outer membrane through an energy consuming mechanism using the proton motive force and the TonB–ExbB–ExbD energy transducing complex from the inner membrane. We solved the crystal structure of the full-length FpvA bound to iron-pyoverdine at 2.7 Å resolution. Signal transduction to an anti-sigma protein of the inner membrane and to TonB–ExbB–ExbD involves the periplasmic domain, which displays a β-α-β fold composed of two α-helices sandwiched by two β-sheets. One iron-pyoverdine conformer is bound at the extracellular face of FpvA, revealing the conformer selectivity of the binding site. The loop that contains the TonB box, involved in interactions with TonB, and connects the signaling domain to the plug domain of FpvA is not defined in the electron density following the binding of ferric pyoverdine. The high flexibility of this loop is probably necessary for signal transduction through the outer membrane.

Preliminary X-ray investigations of several crystal forms of the ferripyoverdine FpvA outer membrane receptor from Pseudomonas aeruginosa bound to ferripyoverdine.

Ferripyoverdine transport across the outer membrane of Pseudomonas aeruginosa by the pyoverdine receptor FpvA and the transcriptional regulation of FpvA involve interactions of the FpvA N-terminal TonB box and signalling domain with proteins from the inner membrane. Several crystallization conditions of FpvA-Pvd-Fe solubilized in C8E4 detergent were obtained and X-ray data were collected from three crystal forms. The resolution limits range from 3.15 to 2.7 angstroms depending on the crystal form. From preliminary analysis of the electron-density maps, the first full-length structure of an outer membrane receptor including a signalling domain should be determined.

Characterization of TonB Interactions with the FepA Cork Domain and FecA N-terminal Signaling Domain

The mechanism of TonB dependent siderophore uptake through outer membrane transporters in Gram-negative bacteria is poorly understood. In an effort to expand our knowledge of the interaction between TonB and the outer membrane transporters, we have cloned and expressed the FepA cork domain (11-154) from Salmonella typhimurium and characterized its interaction with the periplasmic C-terminal domain of TonB (103-239) by isotope assisted FTIR and NMR spectroscopy. For comparison we also performed similar experiments using the FecA N-terminal domain (1-96) from Escherichia coli which includes the conserved TonB box. The FepA cork domain was completely unfolded in solution, as observed for the E. coli cork domain previously [Usher et al. (2001) Proc Natl Acad Sci USA 98, 10676-10681]. The FepA cork domain was found to bind to TonB, eliciting essentially the same chemical shift changes in TonB C-terminal domain as was observed in the presence of TonB box peptides. The FecA construct did not cause this same structural change in TonB. The binding of the FepA cork domain to TonB-CTD was found to decrease the amount of ordered secondary structure in TonB-CTD. It is likely that the FecA N-terminal domain interferes with TonB-CTD binding to the TonB box. Binding of the FepA cork domain induces a loss of secondary structure in TonB, possibly exposing TonB surface area for additional intermolecular interactions such as potential homodimerization or additional interactions with the barrel of the outer membrane transporter.

Crystal Structure at High Resolution of Ferric-pyochelin and its Membrane Receptor FptA from Pseudomonas aeruginosa

Pyochelin is a siderophore and virulence factor common to Burkholderia cepacia and several Pseudomonas strains. We describe at 2.0 A resolution the crystal structure of the pyochelin outer membrane receptor FptA bound to the iron-pyochelin isolated from Pseudomonas aeruginosa. One pyochelin molecule bound to iron is found in the protein structure, providing the first three-dimensional structure at the atomic level of this siderophore. The pyochelin molecule provides a tetra-dentate coordination of iron, while the remaining bi-dentate coordination is ensured by another molecule not specifically recognized by the protein. The overall structure of the pyochelin receptor is typical of the TonB-dependent transporter superfamily, which uses the proton motive force from the cytoplasmic membrane through the TonB-ExbB-ExbD energy transducing complex to transport ferric ions across the bacterial outer membrane: a transmembrane 22 beta-stranded barrel occluded by a N-terminal domain that contains a mixed four-stranded beta-sheet. The N-terminal TonB box is disordered in two crystal forms, and loop L8 is found to point towards the iron-pyochelin complex, suggesting that the receptor is in a transport-competent conformation.

Import of the Transfer RNase Colicin D Requires Site-Specific Interaction with the Energy-Transducing Protein TonB

The transfer RNase colicin D and ionophoric colicin B appropriate the outer membrane iron siderophore receptor FepA and share a common translocation requirement for the TonB pathway to cross the outer membrane. Despite the almost identical sequences of the N-terminal domains required for the translocation of colicins D and B, two spontaneous tonB mutations (Arg158Ser and Pro161Leu) completely abolished colicin D toxicity but did not affect either the sensitivity to other colicins or the FepA-dependent siderophore uptake capacity. The sensitivity to colicin D of both tonB mutants was fully restored by specific suppressor mutations in the TonB box of colicin D, at Ser18(Thr) and Met19(Ile), respectively. This demonstrates that the interaction of colicin D with TonB is critically dependent on certain residues close to position 160 in TonB and on the side chains of certain residues in the TonB box of colicin D. The effect of introducing the TonB boxes from other TonB-dependent receptors and colicins into colicins D and B was studied. The results of these and other changes in the two TonB boxes show that the role of residues at positions 18 and 19 in colicin D is strongly modulated by other nearby and/or distant residues and that the overall function of colicin D is much more dependent on the interaction with TonB involving the TonB box than is the function of colicin B.

The Crystal Structure of the Pyoverdine Outer Membrane Receptor FpvA from Pseudomonas aeruginosa at 3.6 Å Resolution

The pyoverdine outer membrane receptor FpvA from Pseudomonas aeruginosa translocates ferric-pyoverdine across the outer membrane via an energy consuming mechanism that involves the inner membrane energy transducing complex of TonB-ExbB-ExbD and the proton motive force. We solved the crystal structure of FpvA loaded with iron-free pyoverdine at 3.6 angstroms resolution. The pyoverdine receptor is folded in two domains: a transmembrane 22-stranded beta-barrel domain occluded by an N-terminal domain containing a mixed four-stranded beta-sheet (the plug). The beta-strands of the barrel are connected by long extracellular loops and short periplasmic turns. The iron-free pyoverdine is bound at the surface of the receptor in a pocket lined with aromatic residues while the extracellular loops do not completely cover the pyoverdine binding site. The TonB box, which is involved in intermolecular contacts with the TonB protein of the inner membrane, is observed in an extended conformation. Comparison of this first reported structure of an iron-siderophore transporter from a bacterium other than Escherichia coli with the known structures of the E.coli TonB-dependent transporters reveals a high structural homology and suggests that a common sensing mechanism exists for the iron-loading status in all bacterial iron siderophore transporters.

Identification of an Iron-Regulated Hemin-Binding Outer Membrane Protein, HupO, in Vibrio fluvialis : Effects on Hemolytic Activity and the Oxidative Stress Response

In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.

The Solution Structure of the C-terminal Domain of TonB and Interaction Studies with TonB Box Peptides

The TonB protein transduces energy from the proton gradient across the cytoplasmic membrane of Gram-negative bacteria to TonB-dependent outer membrane receptors. It is a critically important protein in iron uptake, and deletion of this protein is known to decrease virulence of bacteria in animal models. This system has been used for Trojan horse antibiotic delivery. Here, we describe the high-resolution solution structure of Escherichia coli TonB residues 103-239 (TonB-CTD). TonB-CTD is monomeric with an unstructured N terminus (103-151) and a well structured C terminus (152-239). The structure contains a four-stranded antiparallel beta-sheet packed against two alpha-helices and an extended strand in a configuration homologous to the C-terminal domain of the TolA protein. Chemical shift perturbations to the TonB-CTD (1)H-(15)N HSCQ spectrum titrated with TonB box peptides modeled from the E.coli FhuA, FepA and BtuB proteins were all equivalent, indicating that all three peptides bind to the same region of TonB. Isothermal titration calorimetry measurements demonstrate that TonB-CTD interacts with the FhuA-derived peptide with a K(D)=36(+/-7) microM. On the basis of chemical shift data, the position of Gln160, and comparison to the TolA gp3 N1 complex crystal structure, we propose that the TonB box binds to TonB-CTD along the beta3-strand.

Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution.

Colicin B (55 kDa) is a cytotoxic protein that recognizes the outer membrane transporter, FepA, as a receptor and, after gaining access to the cytoplasmic membranes of sensitive Escherichia coli cells, forms a pore that depletes the electrochemical potential of the membrane and ultimately results in cell death. To begin to understand the series of dynamic conformational changes that must occur as colicin B translocates from outer membrane to cytoplasmic membrane, we report here the crystal structure of colicin B at 2.5 A resolution. The crystal belongs to the space group C2221 with unit cell dimensions a = 132.162 A, b = 138.167 A, c = 106.16 A. The overall structure of colicin B is dumbbell shaped. Unlike colicin Ia, the only other TonB-dependent colicin crystallized to date, colicin B does not have clearly structurally delineated receptor-binding and translocation domains. Instead, the unique N-terminal lobe of the dumbbell contains both domains and consists of a large (290 residues), mostly beta-stranded structure with two short alpha-helices. This is followed by a single long ( approximately 74 A) helix that connects the N-terminal domain to the C-terminal pore-forming domain, which is composed of 10 alpha-helices arranged in a bundle-type structure, similar to the pore-forming domains of other colicins. The TonB box sequence at the N-terminus folds back to interact with the N-terminal lobe of the dumbbell and leaves the flanking sequences highly disordered. Comparison of sequences among many colicins has allowed the identification of a putative receptor-binding domain.

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.