Exchangeability of N Termini in the Ligand-gated Porins of Escherichia coli*

Daniel C. Scott, Zhenghua Cao, Zengbiao Qi, Matthew Bauler, John D. Igo, Salete M. C. Newton, and Phillip E. KlebbaDagger

From the Department of Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma 73019

Received for publication, December 14, 2000, and in revised form, January 12, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin beta -barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty beta -barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane beta -barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037-1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gram-negative bacteria recognize and transport ferric siderophores (1, 2) through proteins in their outer membrane (OM1). They secrete these chelators in the iron-deficient environments that they encounter in the wild and in the host. Because of its involvement in cellular processes, iron is essential to survival, and bacteria possess efficient systems to obtain it (3-6). However, their competitors in the microbial world parasitize these iron uptake pathways. Bacteriocins and phages bind to siderophore receptors as an initial step in the penetration of the cell wall (7-9). Other microbes are not the only antagonists that target the iron portals of bacteria: synthetic antibiotics that couple antibacterial agents to organic iron complexes also enter through siderophore receptors (10-13). Finally, iron acquisition is crucial to the pathogenesis of Gram-negative bacteria, including Salmonella, Neisseria, Yersinia, Vibrio, and Hemophilus (14-20). In response, higher organisms defend themselves by the sequestration of iron in proteins like ferritin, transferrin, lactoferrin, and conalbumin (21, 22). The competition for iron in vivo is so fierce that one pathogenic species, Borrelia, evolved completely novel biochemical systems that do not require the metal (23).

The OM proteins that initiate the uptake of iron, antibiotics, colicins, and phage function as ligand-gated porins (LGP (24-26)). They contain a transmembrane channel formed by amphiphilic beta -strands that project to the cell surface as large loops (27-29). In this sense the proteins are fundamentally porins in nature (30-32). However, LGP differ from other porins, because they bind the molecules they transport with high affinity and because they contain a globular N terminus that resides within their transmembrane channel (Fig. 1). When ligands adsorb to siderophore receptors, they trigger unknown events that induce their own transport into the cell (33-35). In this sense the receptors are ligand-gated. Finally, LGP transport requires proton motive force (36-38) and the participation of another cell envelope protein, TonB (39-41), which presumably promotes the opening of the closed channels so ligands may enter. The crystal structures of the ferric enterobactin receptor, FepA (27), and the ferrichrome receptor, FhuA (28, 29), defined the architecture of siderophore transporters but did not answer several pressing questions about their biochemical mechanisms.

Their ligand recognition and transport processes, for example, are only vaguely defined. The OM receptors of Escherichia coli discriminate numerous ferric siderophore complexes, which, despite their similar chelation geometry (hexacoordinate) and size (600-1000 Da), are distinct in composition and charge (1, 2, 42). Each ferric siderophore enters through a different OM iron transporter. Ferric enterobactin (FeEnt), the native E. coli siderophore, penetrates through FepA (43), whereas ferrichrome (Fc), a fungal product that E. coli also utilizes, passes through FhuA (44). Does LGP specificity derive from residues in their surface loops or from amino acids in the N-domain, at the entrance to the membrane channel (Fig. 1)? Experiments on a FhuA mutant devoid of its N-terminal domain (45) were relevant to this issue, as well as that of ligand transport. In those studies the N-domain of FhuA was not necessary for Fc, colicin, and bacteriophage uptake. The experiments we report herein duplicated and confirmed those findings on FhuA and characterized comparable constructions from FepA, which gave almost identical results. Furthermore, when we genetically exchanged the N termini of FepA and FhuA, both hybrid proteins still bound and transported their appropriate ligands.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Bacterial Strains, Plasmids, and Media-- Bacteria harboring plasmids (Table I) carrying the genes of interest were cultured in LB broth or MOPS minimal media (46). It was impractical to create single-copy chromosomal derivatives of the numerous constructs we generated. Instead, we individually transferred all of the genes, including wild type fepA and fhuA, to the low-copy plasmid pHSG575, which exists in E. coli at a level of two to three copies/cell (47).

                              
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Table I
Strains and plasmids

Genetic Engineering-- We used PCR to join the FepA N terminus (residues 1-152) to the FhuA C terminus (residues 160-723), and the FhuA N terminus (residues 1-155) to the FepA C terminus (residues 149-724). We first cloned the N- and C-terminal domains of fepA and fhuA and then ligated them together. For instance, for FepNFhubeta , we PCR-amplified the DNA encoding FepN, incorporating PstI and BamHI sites in the forward and reverse primers, respectively, and inserted the product into the low copy plasmid pHSG575 (47). We then PCR-amplified the DNA encoding Fhubeta , flanked by BamHI and SacI restriction sites, and ligated it to FepN DNA in pHSG575. Additionally, we employed oligonucleotide-directed mutagenesis (QuikChange, Stratagene, San Diego, CA) to delete residues 17-150 of fepA (creating Fepbeta ), residues 3-150 of fepA (Fepbeta 2), and residues 5-160 of fhuA (Fhubeta ). Except when noted, fepA, fhuA, and their derivatives were all analyzed on pHSG575, which was used for binding, nutrition, and transport experiments.

Binding and Transport-- The adsorption of [59Fe]Ent and [59Fe]Fc was measured with metabolically inactive KDF541 (46) expressing FepA or FhuA, respectively. [59Fe]Ent and [59Fe]Fc were prepared at a specific activity of ~200 cpm/pM and chromatographically purified. Binding manipulations were performed at 0 °C. A mid-log bacterial culture was chilled on ice for 1 h, and an aliquot (containing ~5 × 107 cells) was pipetted into a 50-ml test tube and incubated on ice. 25-ml volumes of ice-cold MOPS minimal media, containing varying concentrations of [59Fe]-siderophore, were poured into the tubes to achieve rapid and thorough mixing. After 1 min the binding reactions, which were performed in triplicate, were filtered through 0.45-µm nitrocellulose, the filters were washed with 10 ml of 0.9% LiCl and counted in a Packard Cobra gamma counter. The initial adsorption reaction reached equilibrium within 5 s at physiological temperatures and within 1 min on ice. The FepA-deficient strain KDF541 was simultaneously tested as a negative control, and any nonspecific adsorption of [59Fe]siderophores by this strain was subtracted from the experimental samples. Whenever necessary because of low cpm bound, the assay samples were counted for an extended period of time (up to 30 min) to decrease standard error. Binding data were analyzed using the Bound versus Total equation of Grafit 4.013 (Erithacus Ltd., Middlesex, UK).

Ferric siderophore uptake was qualitatively evaluated by nutrition tests (46), and the transport of [59Fe]Ent and [59Fe]Fc was quantitatively measured in live bacteria (46). For ferric siderophore acquisition (and general and specific porin-mediated transport as well (48)), the outer membrane transport stage constitutes the rate-limiting step of the overall uptake process (4, 6, 24, 26, 46, 49-51). Periplasmic binding proteins (51) and the components of the inner membrane permease complexes are either constitutively expressed or de-repressed by iron starvation. Therefore, our transport data describe the outer membrane components of the process, FepA, FhuA, and their mutant derivatives.

Transport manipulations were performed at 37 °C. A volume of mid-log bacterial culture (50-100 µl containing ~5 × 107 cells) was pipetted into a 50-ml test tube and incubated in a 37 °C water bath. Without delay, 25 ml of prewarmed MOPS minimal media, containing glucose (0.2%), appropriate nutritional supplements, and varying concentrations of [59Fe]siderophores, were poured into the tube to achieve rapid and thorough mixing. The transport reactions were quenched by the addition of a 1000-fold excess of nonradioactive ferric siderophores, immediately filtered through 0.45-µm nitrocellulose, and the filters were washed with 10 ml of 0.9% LiCl and counted in a Packard Cobra gamma counter. Kinetic parameters were determined from the initial rates of uptake, which were calculated at each substrate concentration from two independent measurements made in triplicate at 5 and 15 s: cpm bound to the cells at 5 s were subtracted from the cpm associated with the cells at 15 s (10-s uptakes). For some mutants with low uptake rates (FepNFhubeta , FhuNFepbeta , Fepbeta ) we extended the uptake period to 60 min (6). In all transport experiments, the FepA-deficient strain KDF541 was simultaneously tested as a negative control, and any nonspecific adsorption of [59Fe]Ent by this strain was subtracted from the experimental samples. Transport results were analyzed according to the Michaelis-Menten equation, using Grafit 4.013.

Bacteriocin and Bacteriophage Susceptibility-- Serial dilutions of colicins B, D, or M, and bacteriophage T5 and phi 80 were prepared in LB broth in microtiter plates, and 5-µl volumes of the dilutions were transferred to LB plates seeded with the strain of interest, using a Clonemaster (Immusine Corp., San Leandro, CA). The titer of the phage and colicins was expressed as the reciprocal of the highest dilution that cleared the bacterial lawn.

SDS-PAGE and Western Immunoblots-- Proteins were separated on SDS-PAGE gels (52), and either stained with Coomassie Blue or electrophoretically transferred to nitrocellulose paper. Western immunoblots were incubated with anti-FepA mAbs 26 or 45 (52), developed with 125I-protein A (49), quantitated by image analysis with a Packard Instant Imager, and visualized by exposure of x-ray films. The former antibody recognizes an epitope in the N-terminal globular domain of FepA (bounded by residues 27and 37 (52)); the latter binds in loop 4 of the C-terminal barrel domain, near residue 329 (49, 52). For native gel electrophoresis, 50 µg of OM or 2 µg of purified protein was resuspended in LDS sample buffer and sonicated in an ice water bath for 5 min. After a 1-min centrifugation in a microcentrifuge, the samples were electrophoresed overnight at 5 mA and 4 °C on LDS-PAGE gels (53).

Mutant Protein Expression and Localization-- The expression of FepA and its mutants was quantitated by Western immunoblots of whole cell lysates with anti-FepA monoclonal antibodies 26 and 45 and 125I-protein A. Protein concentrations were determined by image analysis, relative to standards. The localization of the chimeric proteins was determined by fractionation of inner and outer membranes on sucrose gradients (54), followed by Western immunoblots of the fractions.

Enzyme-linked Immunosorbent Assays (ELISA)-- Purified OM fragments or purified, denatured FepA, were suspended in 10 mM ammonium acetate, 10 mM ammonium carbonate, pH 8.3, at 10 and 0.5 µg/ml, respectively, dispensed into microtiter plates (Immulon), and incubated at 4 °C overnight. All further incubation steps were performed at 25 °C. In the morning, excess or unabsorbed antigen was removed by three washes with Tris-buffered saline containing 0.05% Tween 20, pH 7.4 (TBS-Tween), and the plates were blocked with 2% bovine serum albumin in TBS for 30 min. The plates were washed three times with TBS-Tween, and serial dilutions of anti-FepA mAbs in 50 µl of blocking buffer were added and incubated for 1 h. The blocking buffer was removed and 50 µl of a 1/100 dilution of goat-anti-mouse immunoglobulin-alkaline phosphatase (Sigma Chemical Co.) in blocking buffer was added to the plates. After incubation for 1 h at 25 °C, the plates were washed three times and developed with 50 µl of p-nitrophenyl phosphate (1 mg/ml, Sigma). After a 1-h incubation at 25 °C, 50 µl of 2 N NaOH was added to stop the reaction and absorbance was measured at 405 nm with a microplate reader.

OM Protein Cross-linking-- Bacteria grown in MOPS minimal media or sucrose gradient-purified OM fractions were suspended at 109 cells/ml or 10 mg/ml, respectively, in 4 mM SulfoEGS, a water-soluble homobifunctional cross-linker (Mr 661 Da) that preferentially reacts with primary amines, for 2 h at 0 °C. The compound contains a 16-Å spacer arm, which is cleaved by reaction with hydroxylamine. After cross-linking, cells or OM proteins were solubilized in sample buffer, subjected to SDS-PAGE, and stained with Coomassie Blue. When indicated, 5 µM ferric enterobactin was added to the cells prior to cross-linking. Cross-linked bands were excised from the gels, cleaved with hydroxylamine, electroeluted, and re-electrophoresed: The identity of the cross-linked proteins was determined by sequence analysis of their N-terminal 15 residues (Protein and Nucleic Acid Sequence Facility, Medical College of Wisconsin).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genetic Exchange of LGP N Termini-- To understand the basis of ligand specificity in FepA and FhuA, we switched their N-terminal domains and biochemically characterized the resulting hybrid proteins. Although Braun et al. (45) reported the ability of an N-terminal deletion of FhuA to transport, they did not consider the affinity of the mutant for its ligands, nor the kinetic parameters of the uptake process. Our experiments duplicated their construction, created comparable deletion mutants of FepA, and created the two chimeric proteins that switched the N-domains of FepA and FhuA. In the latter case, the protein engineering conserved three structural features of FepA and FhuA: the globular N-domain, the beta -barrel of the C-domain, and the beta -turn that joins them (Fig. 1). The resulting clones avoided the deletion or introduction of amino acids in the junction sequence: FepNFhubeta contained the N terminus and beta -turn of FepA connected to the beta -barrel of FhuA; FhuNFepbeta contained the N terminus of FhuA linked to the beta -turn and beta -barrel of FepA.


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Fig. 1.   Crystal structures of FepA (top left), FhuA (top right), and the chimeric constructions FhuNFepbeta (bottom left) and FepNFhubeta (bottom right). The N-terminal domains of FepA and FhuA are red and orange, their beta -barrels are green and blue, and the beta -turns between these regions are cyan and magenta, respectively. The junction regions are also shown in space filling representations, including Arg and Lys (green), Ser and Thr (white), and Glu (cyan); Pro and Gly are gray; Trp is purple.

The hybrid proteins functioned with unexpected efficiency and selectivity, conferred by the beta -barrels and their loops. The N-terminal domains did not affect the discrimination of ferric siderophores. FhuNFepbeta bound FeEnt but not Fc, whereas FepNFhubeta bound Fc but not FeEnt. FepA adsorbs FeEnt with subnanomolar affinity, which persevered in the FhuNFepbeta chimera: The Kd of its binding reaction with FeEnt was 0.2 nM (Fig. 2, Table II). The reverse hybrid (FepNFhubeta ) bound Fc with equivalent affinity to that of wild type FhuA: The Kd of its binding reaction with Fc was 0.6 nM (Fig. 2, Table II). So, despite their heterologous N termini, the chimeras maintained the selectivity of their beta -barrels, with the affinity of wild type receptors.


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Fig. 2.   FeEnt and Fc binding. The adsorption of [59Fe]Ent (open symbols) and [59Fe]Fc (filled symbols) was measured with metabolically inactive KDF541 expressing FepA or FhuA (open circle ), FhuNFepbeta (triangle ), FepNFhubeta (down-triangle), Fepbeta (), and Fhubeta (diamond ), carried on pHSG575.

                              
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Table II
Biochemical properties of FepA and FepA mutants

The constructs that deleted the N-domains of FepA and FhuA (Fepbeta and Fhubeta ) also bound ferric siderophores with wild type affinities (Fig. 2 and Table II). Our Fhubeta construct was identical in composition to that of Braun et al. (45). Regarding the beta -barrel-only clones of FepA, we engineered two different derivatives. In fepADelta 17-151 (Fepbeta ) we removed most of the the N-domain but left amino acids 1-16, which include the TonB-box region, fused to Gly-151. Besides Fepbeta we engineered fepADelta 3-151 (Fepbeta 2), which eliminated the TonB box. Except for a slight difference in expression (Fepbeta was better), the two clones functioned identically in binding, nutrition, and transport assays; we only report data for Fepbeta . The empty beta -barrels of FhuA and FepA avidly bound their appropriate ferric siderophore, indicating that the surface loops select the iron complexes that the bacteria encounter in their environments: the loops identify correct iron chelates and reject improper ones. Although they were irrelevant to ferric siderophore recognition, the N-terminal domains of FepA and FhuA were needed for optimum binding. Bacteria-expressing constructions that exchanged or deleted them adsorbed ferric siderophores with much lower capacities (Fig. 2 and Table II). This reduction did not result from poor expression (Table II), improper localization, or instability (Fig. 4): rather, biochemical characterizations indicated that only a fraction (~10%) of the mutant proteins bound ferric siderophores. Thus, a correct N-domain, which was not needed for discrimination of the metal chelates, was essential for their maximal binding. Without their homologous N-domains, mutants of FepA and FhuA adsorbed much lower amounts of ferric siderophores to the cells.

Interactions with Colicins and Bacteriophage-- The beta -barrels of the mutant proteins also dictated interactions with bacteriocins and bacteriophages. The FepA beta -barrel conferred susceptibility to colicins B and D, but not M or the phages that use FhuA, again demonstrating the irrelevance of the N-domain to ligand selection. Likewise, the FhuA barrel recognized T5 and phi 80, and the transposition of the FepA N terminus into it did not change this specificity (Table II). So, the antagonism between iron chelates and noxious agents for adsorption to FepA and FhuA derives from competition for structural elements in their beta -barrels and loops, in accord with the prior localization of residues in FepA that are needed for efficient colicin reception. Yet, the exchange of N termini reduced the efficiency of colicin B and D killing, suggesting that these bacteriocins further interact with the globular domain during the implementation of their toxicity. Unlike Braun et al. (45), we could not detect the ability of Fhubeta to confer susceptibility to colicin M. This discrepancy may stem from the low activity of our colicin M preparation.

The N-domain in Transport-- The occlusion of the FepA and FhuA channels by their own N termini raises another question: What is the function and structural disposition of the N-domain during transport? Passage of ferric siderophores requires a minimum diameter of about 15 Å, which does not exist in either FepA or FhuA. Does the N-terminal domain exit the beta -barrel during metal uptake, or change shape within the pore to create a sufficient opening for ligand transit? The former mechanism faces an energetic barrier, because the crystal structures of FepA and FhuA describe many residues in appropriate positions for hydrogen bonds between the N-domains and barrel walls. The latter notion, that the conformation of the N terminus temporarily changes to create a pore, appears more plausible, but transient channels of such magnitude have not been observed in membrane proteins. Furthermore, although colicins generally comprise a slender, elongated shape, their entry into or passage through even an open porin channel faces severe steric obstacles (55).

Uptake experiments with the mutant proteins, especially those containing the FhuA barrel, bore relevance to these questions. Despite its binding ability, the presence of the FepA N terminus within it made it unlikely for FepNFhubeta to transport Fc. Yet the chimera efficiently internalized the hydroxamate siderophore. Fc stimulated the growth of bacteria expressing FepNFhubeta as much as strains producing FhuA (Fig. 3A), in a TonB-dependent manner. Quantitative determinations explained this result: despite a slightly lower overall uptake affinity for Fc than FhuA (Km values of 2.5 and 0.6 nM, respectively; Fig. 2 and Table II), the chimeric protein displayed a comparable turnover number to the wild type protein (k3 values of 6.5/min and 4/min, respectively; Table II). Furthermore, the isolated FhuA beta -barrel transported Fc with similar affinity (Km = 3.6 nM) and at a faster rate (k3 = 8.3/min). Thus neither was an N-domain required for Fc uptake, nor did the introduction of the FepA N terminus into the FhuA barrel impair the internalization reaction: the 160 amino acids of the globular domain were superfluous to Fc transport. Mutant proteins containing the FepA beta -barrel were less efficient, but they corroborated the dispensability of the N terminus to transport: Both FhuNFepbeta and Fepbeta conferred TonB-dependent FeEnt uptake, albeit slower than wild type FepA (k3 values of 0.9/min, 0.3/min, and 6.4/min, respectively; Fig. 3 and Table II). The low transport rates of FhuNFepbeta and Fepbeta explained their marginal abilities in nutrition tests (Fig. 3a). All of the plasmids failed to support growth in KDF571 ((24) tonB; Fig. 3). We also constructed and analyzed a second, similar chimera, FhuNFepbeta 2, containing the N terminus and turn of FhuA linked to the barrel of FepA, whose biochemical properties were equivalent to those of FhuNFepbeta (data not shown). Accuracy was not compromised in the binding and transport measurements of strains with low capacity (FepNFhubeta , FhuNFepbeta , Fepbeta , and Fhubeta ). The 59Fe binding and uptake assays were sufficiently sensitive to quantitate even slight binding relative to the negative controls, which are absolute (46, 49). The close conformity of the data to nonlinear fit calculations from the Bound versus Total and Michaelis-Menten equations (Figs. 2 and 3, insets) substantiated the accuracy of the measurements. For bacteria expressing FepNFhubeta , FhuNFepbeta , and Fepbeta , the samples with the lowest capacities, the mean standard error of affinity measurements (Kd and Km) was 9.5%. For the same strains the mean standard error of capacity and Vmax measurements was 2.3%.


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Fig. 3.   FeEnt and Fc transport. Top, ferric siderophore uptake was qualitatively evaluated by nutrition tests. All of the plasmids failed to support growth in the tonB strain KDF571, producing tests identical to that of KDF541. Bottom, the transport of [59Fe]Ent (open symbols) and [59Fe]Fc (filled symbols) was quantitated in live bacteria. Strains and their symbols are the same as in Fig. 2, except (inset) uptake by KDF571 (tonB) harboring pFepbeta (x) and pFhuNFepbeta (asterisks).

Conformation of the Hybrid Proteins-- The tertiary structures of the mutant proteins were pertinent to the understanding of their binding and transport abilities. We characterized the conformations of the chimeric proteins by native electrophoresis, immunochemistry, and nonspecific permeability. The beta -barrels of OM proteins are resistant to denaturation by SDS, and they have a compact native structure that imparts enhanced mobility in PAGE (56, 57). Denaturation of OM proteins by heating in ionic detergents eliminates this globular structure, reverting the abnormal electrophoretic behavior. In their native states FepA and FhuA exhibit the same compact shape and increased electrophoretic migration (52, 58), but they are less stable than general or specific porins (59), and therefore more sensitive to denaturation. For these reasons, electrophoretic behavior was a revealing measure of the tertiary structure of the chimeric proteins. When electrophoresed in LDS at 4 °C, FhuNFepbeta and FepNFhubeta displayed the same rapid mobility as FepA and FhuA, respectively (Fig. 4), intimating that all four proteins possessed a similarly compact globular structure.


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Fig. 4.   Expression, localization, and nondenaturing electrophoresis of mutant proteins. A, expression. Western immunoblots stained with anti-FepA mAbs 26 (top) and 45 (bottom) and developed with 125I-protein A. Lanes 1-3, 1, 3, and 5 µg of purified FepA, respectively; lanes 4-10 each contain lysates of 2. 5 × 108 bacterial cells, except lane 9, which contained 108 cells. Lane 4, KDF541 (fepA, fhuA); 5, KDF541/pFepNFhubeta ; 6, KDF541/pFhuNFepbeta ; 7, KDF541/pITS23 (fepA+); 8, BN1071 (fepA+, fhuA+); 9, KDF541/pITS449 (fepA+); 10, KDF541/pFepbeta . Expression levels were quantitated by image analysis and tabulated in Table II. B, localization. Inner and outer membranes from bacteria expressing the chimeric proteins were fractionated on sucrose gradients, and 30 µg of each sample was analyzed by Western immunoblots with monoclonal antibodies 26 (top) and 45 (bottom). Lane 1, KDF541 cell lysate; lanes 2-4, KDF541/pITS23 lysate, IM and OM, respectively; lanes 5-7, KDF541/pFhuNFepbeta lysate, IM and OM; lanes 8-10, KDF541/pFepNFhubeta lysate, IM and OM. C, Nondenaturing LDS-PAGE. The left panel is a Coomassie Blue-stained, nondenaturing LDS-PAGE gel; the right panel is a Western immunoblot of a nondenaturing LDS-PAGE gel, stained with a mixture of anti-FepA mAbs 26 and 45 and developed with 125I-protein A. Lane 1, molecular mass markers (Bio-Rad, Hercules CA); lanes 2 and 3, purified, denatured FepA and FhuA, respectively; lanes 4 and 5, purified, nondenatured FepA and FhuA, respectively; lanes 6-8, OM fragments (30 µg) from KDF541 expressing FepA, FhuNFepbeta , and FepNFhubeta .

As a further indication of the folding of FepNFhubeta , we determined the accessibility of epitopes within its (FepA) N terminus to mAb binding. In ELISAs utilizing four different anti-FepA mAbs that bind epitopes in the N-terminal 150 residues, FepNFhubeta was indistinguishable from wild type FepA (Fig. 5). The antibodies against the N-domain did not react with either protein unless the antigen was denatured before adsorption to the plates, suggesting that the N-domain of the chimeric protein was sequestered within Fhubeta , in a comparable manner to the N-domain within the barrel of the wild type protein. The unavailability of antibodies to the FhuA N terminus2 precluded a similar analysis of FhuNFepbeta . However, we cytofluorimetrically assessed its proper folding, with anti-FepA mAbs against surface epitopes in the loops of its (FepA) C-terminal domain. Like FepNFhubeta in ELISA, FhuNFepbeta was indistinguishable from wild type FepA in flow cytometry (Table II).


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Fig. 5.   Analysis of FepNFhubeta by ELISA. Outer membranes from KDF541 carrying pITS23 (A) or pFepNFhubeta (B), or purified, denatured FepA (C) were adsorbed to microtiter plates and tested with anti-FepA mAbs 1 (open circle ), 12 (), 27 (), 41 (triangle ), 33 (black-diamond ), and 45 (black-down-triangle ), or normal mouse serum (x). The first four antibodies recognize different epitopes in the N-terminal globular domain, whereas the latter two bind epitopes in surface loops of the barrel. A and B plot mean values from three experiments; C shows the results of a single experiment.

Finally, neither chimeric protein conferred increased permeability to large antibiotics, whereas bacteria expressing either Fepbeta or Fhubeta showed enhanced susceptibility to bacitracin (1500 Da; Table II), as expected for OM proteins containing a large, open channel. In total, the three different approaches all suggested that both hybrid proteins assembled with N termini lodged within their heterologous beta -barrels.

Cross-linking with SulfoEGS-- In the presence of formaldehyde, FepA cross-links to another cell envelope protein, TonB (for review see Ref. 4). However, skepticism about formaldehyde as an indicator of proximity led us to test other cross-linking agents with more specific chemical targets. The bifunctional, cleavable cross-linking agent SulfoEGS selectively reacts with primary amines and is too large (661 kDa) to pass through the aqueous channels of the OM, restricting its chemical action to Lys residues on the membrane surface. When applied to live bacteria or purified Escherichia coli outer membranes, SulfoEGS generated two prominent protein complexes that included FepA, of approximate molecular masses 100 (band 1) and 120 kDa (band 2; Fig. 6). The identity of the cross-linked protein(s) in the 100-kDa complex is currently unknown. When cleaved, the 120-kDa product yielded, in addition to FepA, the major OM proteins OmpF/C and OmpA, as determined by Edman degradations of their N-terminal 15 residues (Fig. 6). The ostensible discrepancy in the concentration of FepA, OmpF/C and OmpA in lane 9 of Fig. 6 occurs because the molecular mass of FepA (81 kDa) is almost 3-fold higher than those of OmpF/C or OmpA. Accordingly, in SDS-PAGE of a (dissociated) 1:1 complex of FepA·OmpA, for example, a nearly 3-fold lower intensity is expected for the OmpA band. Two conditions influenced the cross-linking reaction between FepA and the major OM proteins. First, both in vivo and in vitro the binding of FeEnt to FepA eliminated band 2, and drastically reduced the level of band 1. The removal of the N-domain, in Fepbeta , produced analogous results, reducing the levels of the 100- and 120-kDa products to barely detectable levels (Fig. 6).


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Fig. 6.   Cross-linking of bacteria with SulfoEGS. A, outer membranes from BN1071 (lanes 1-3) or KDF541/pITS449 (lanes 4-6) were incubated with the cleavable cross-linker Sulfo-EGS, subjected to SDS-PAGE, and stained with Coomassie Blue. Lanes 1 and 4, no cross-linking; lanes 2 and 5, cross-linked by SulfoEGS. Lanes 3 and 6, cross-linked by SulfoEGS in the presence of 5 µM FeEnt. Lane 7, molecular mass standards myosin (200 kDa), beta -galactosidase (116 kDa), phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), and carbonic anhydrase (31 kDa). Lane 8, purified FepA. Lane 9, band 2 in lane 2 was excised, cleaved with hydroxylamine, electroeluted, and re-electrophoresed: Note the resulting proteins that migrate with apparent molecular masses of 34 and 36 kDa, whose identity as OmpF/C and OmpA was verified by sequence analysis of their N-terminal 15 residues (data not shown). B, outer membranes from KDF41/pITS23 (lanes 1-3), or live cells of KDF541 harboring pITS23 (lanes 4-6) or pFepbeta (lanes 7-9) were either untreated (lanes 1, 4, 7), or cross-linked with Sulfo-EGS in the absence (lanes 2, 5, 8) or presence of 5 µM FeEnt (lanes 3, 6, 9), and cell lysates were transferred to nitrocellulose and analyzed by immunoblot with anti-FepA mAb 45, developed with 125I-protein A.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Since its discovery (39, 40), the TonB dependence of ferric siderophore acquisition has remained obscure and controversial. The crystal structures of FepA and FhuA did not explain this aspect of ligand uptake, although a change in the structure of the N-terminal extremity of FhuA during Fc binding (28, 29) raised the possibility of conformational signaling between the receptor protein and TonB, or another component of the transport system. The localization of the "TonB-box" of siderophore transporters within the region of the N terminus that changed conformation in response to ligand binding was superficially consistent with this notion of signal transduction between the bound receptor protein and TonB. In this light it was unexpected that a mutant FhuA protein devoid of the N-terminal globular domain effectively transported Fc (45). Our experiments had several objectives relevant to this phenomenon: (i) to verify the phenotype of the N-domain deletion of FhuA, (ii) to determine whether equivalent mutants of FepA transport FeEnt, (iii) to consider the functional exchangeability of the FepA and FhuA N termini, and (iv) to provide thermodynamic and kinetic descriptions of the binding and transport reactions by such mutant receptors.

Equilibrium binding studies unequivocally demonstrated that the specificity of LGP, and their high affinity for ferric siderophores, derive from their surface loops. The abilities of both Fepbeta and Fhubeta to correctly discriminate FeEnt and Fc, and to bind them with wild-type affinity, confirms this point. These data also concur with the identification of aromatic residues in the exterior-most portion of the loops around the entrance to the FepA vestibule, that function in FeEnt recognition (49). In the crystal structure of FhuA, Fc lodged deep within the vestibule interior, in contact with residues at the top of the globular domain, suggesting that the N-domain contributes to the overall efficacy and specificity of the ligand binding process. However, the subnanomolar binding affinity of the two proteins that lack the N-domain, Fhubeta (for Fc) and Fepbeta (for FeEnt), indicate that in both receptor proteins the globular domain does not significantly affect ligand recognition or binding. The correct adsorption of colicins and phages by both chimeras reiterates that, if the N-domain acts at all in the binding reaction, then it's contributions are minimal and secondary to those of the surface loops.

One of the mutant constructs (FhuNFepbeta ) had a lower expression level, and others conferred lower binding capacities than wild type FepA. To compare the uptake rates of the strains, it was therefore necessary to determine the number of molecules of ligand transported per receptor protein per unit of time. k3 describes this rate, in essence the turnover number of each of the transport proteins. It derives from the simple Michaelis-Menten description of the siderophore uptake reaction as follows.
<UP>   FeX<SUB>out</SUB></UP>+<UP>receptor</UP> <LIM><OP><ARROW>⇄</ARROW></OP><LL>k<SUB>2</SUB></LL><UL>k<SUB>1</SUB></UL></LIM> <UP>FeX-receptor</UP> <LIM><OP><ARROW>→</ARROW></OP><UL>k<SUB>3</SUB></UL></LIM> <UP>receptor</UP>+<UP>FeX<SUB>in</SUB></UP> (Eq. 1)
k3 is the only measure available by which to compare, in vivo, populations of proteins with different capacities and concentrations in the membrane. In our study k3 is exactly equivalent to the term kcat, the recommended basis for rate comparisons of enzymes: k3 = Vmax/Cap; kcat = Vmax/ET.

Besides its high affinity, Fhubeta transported Fc in a TonB-dependent manner, with a better turnover number than wild type FhuA (k3 in Table II). Although the comparable FepA derivatives had reduced transport rates, their activities must be related to the following series of controls: fepA bacteria, tonB bacteria, and fepA+, energy-depleted bacteria. These control strains/conditions show absolute inability to transport any of the relevant ligands (Table II and Fig. 3 (see also Refs. 24, 46, 49, 50)). Therefore, even the least active construct, FhuNFepbeta , showed the complete range of functionalities. These data lead to the same conclusion as that reached by others (45): The N termini of FhuA and FepA are not needed for the transport of Fc and FeEnt. Besides their importance to the understanding of ferric siderophore transport (see below), these data demonstrate that TonB does not function by an interaction with the N-terminal 150 amino acids of ferric siderophore receptors, despite indirect evidence from genetic suppression (60-62), cross-linking (63-68), and affinity chromatography (69). Thus the concepts of transmembrane signaling and/or functional interactions between LGP and TonB, through the TonB-box, are specious. Instead, the results suggest the contrary, that, subsequent to the binding of metal chelates, the surface loops of LGP undergo TonB-dependent conformational changes during the transport of metal chelates. This conclusion from genetic engineering concurs with biophysical studies of loop conformational dynamics in vivo (25).

Also contrary to previous suppositions (27-29, 70) the globular domain apparently does not create a siderophore-specific transit pathway to the periplasm. The idea of a substrate-customized channel conflicts with the efficient internalization of the neutral Fc iron complex by FepNFhubeta , which contains an N-domain adapted to a negatively charged, catecholate ferric siderophore. The heterologous N-domain of FepNFhubeta did not impair Fc uptake: like Fhubeta , the chimera transported at a faster rate than wild type FhuA. The FeEnt transport activity of FhuNFepbeta reiterated this point. The ability of the chimeras to internalize the ferric siderophore that their beta -barrel domains adsorbed undercuts the notion of a transient, ligand-specific pathway to the periplasm. On the contrary, bound metal chelates enter through a nonspecific route, neither created nor regulated by the N terminus.

If it does not act in solute recognition or uptake, then what is the function of the N terminus? Constructs without an N-domain, or with an aberrant one, adsorbed ferric siderophores to much lower capacities than the wild type receptors, suggesting that in the native proteins the proper insertion of the N-domain into the barrel facilitates binding. Such an effect may occur by protein·protein interactions between the globular domain and the barrel, which optimize the conformation of surface loops. In the populations of empty beta -barrel proteins that we studied only a fraction (~10%) of the molecules bound ferric siderophores. However, the percentage of receptor proteins that adopted an appropriate conformation, perhaps by random conformational motion (the flexibility of the loops of FepA was apparent in its x-ray structure determination), attained wild type affinity. So, despite the fact that it does not act in ligand discrimination, the globular domain enhances the binding capacity of ferric siderophore receptors. In this sense though, the N-domains of FepA and FhuA were not interchangeable, because like Fepbeta and Fhubeta the chimeric receptors also showed reduced binding capacity. We addressed the possibility that in FepNFhubeta and FhuNFepbeta the globular domains do not enter the porin beta -barrels but remain suspended in the periplasm. Electrophoretic and immunochemical characterizations argued against this alternative view of their structure: Both hybrid proteins were identical to wild type FepA and FhuA in these tests. It is therefore likely that the globular domains of the chimeras reside within their beta -barrels, but the absence of appropriate interactions between the heterologous N- and C-domains prevents the attainment of wild type binding capacity.

In addition to its function in the optimization of ferric siderophore binding, the presence of the globular domain within the beta -barrel may preserve the integrity of the cell envelope to noxious small molecules. The bacterial OM creates a selective permeability barrier that permits uptake of nutrients and vitamins, generally smaller in mass than 600 Da, and excludes larger, toxic, hydrophobic molecules, including the natural detergents of the animal gut (48). In this light the obstruction of the large LGP channel, which when open renders the bacteria susceptible to bile salts in the gut, is an important rationale for the arrangement of the binding-receptive state that we propose. This general trend of OM protein design appears in all other structurally characterized porins, including the E. coli transporters OmpF and LamB, which both contain a uniquely oriented loop (L3) that restricts permeability through their channels (30-32).

The crystal structure of FhuA created controversy about potential conformational changes that may occur in the surface loops of LGP during ferric siderophore binding and transport. Although ample evidence from biophysical studies suggested structural dynamics within FepA (25, 33, 71, 72) and FhuA (34) the absence of different loop conformations in the crystals of ligand-bound and ligand-free FhuA challenged this deduction. Nevertheless, it is most relevant to consider conformational change in the in vivo environment, free from the constraints of crystallography. A crystal structure captures only one of the many conformational states that a protein may adopt, as aptly stated by Gutfreund: " ... from a picture of a racehorse, you can't tell how fast it can run" (73). The cross-linking experiments reported herein provide further contradictions to the static picture surmised from crystallography. When live bacteria were exposed to sulfoEGS, FepA cross-linked to the major OM proteins OmpF/C and OmpA. Binding of FeEnt eliminated the cross-linking, as did the removal of the N-domain, in Fepbeta . The experiment demonstrates two loop conformations in vivo: an unoccupied form in which the surface loops reach close proximity to contiguous OM proteins, and a bound form with closed loops that do not cross-link to other proteins. The failure of Fepbeta to cross-link to OmpF/C and OmpA suggests the need for the N-domain to enter the barrel to create the open state.

We've previously objected to the use of formaldehyde cross-linking in the study of proposed physical interactions between TonB and FepA, because of a lack of control data on the cross-linking of TonB to non-TonB-dependent OM proteins, uncertainties about the nature and identities of chemical targets of the formaldehyde-activated residues within the cross-linked proteins, and the absence of quantitation of the very small amount of total cross-linked products (4). The cross-linking experiments we report attempted to satisfy these same concerns, by employing a reagent with well defined targets and methods (Coomassie Blue-stained SDS-PAGE gels and 125I-protein-A immunoblots) that were amenable to the identification of all detectable cross-linked products and their precise quantitation. In our experiments up to 70% of the total FepA in the OM became involved in cross-links. Band 2 (Fig. 6) was abundant enough that we excised it from gels and determined the N-terminal sequences of its components.

The several experimentally defined forms of LGP provide insight into the iron transport process. First, structural differences exist between the unbound and bound receptor protein, implicating conformational change as a main element of the ligand binding process. During ferric siderophore acquisition LGP likely progress between multiple conformations; at least one of the transitions between structural forms requires energy and/or the participation of TonB. According to this view, FepA initially assumes an "open" state, with the N-domain inside the barrel, where it compels the unoccupied loops to an extended, optimized configuration for binding. FeEnt adsorption to the loops stimulates their coalescence, creating a "closed" complex. Besides our cross-linking data, ESR spectroscopy (33, 72) described these open and closed forms, and kinetic fluorescence analyzes (71) showed the movement of FeEnt between two distinct sites during binding. Consistent with this view, site-directed mutagenesis defined residues in an exterior binding site (Tyr-272, Phe-329) and an interior site (Arg-316 (50) and Tyr-260 (49)). After the ferric siderophore attains binding equilibrium, it sits poised above the globular N-domain, itself situated within the channel. Crystallographic experiments presumably captured and described such a closed complex of FhuA. The transport-competent, empty beta -barrels of FhuA and FepA constitute another noteworthy form of LGP. Their very existence and functionality implies that the N-domain exits the barrel as ligands traverse the OM. On this basis we propose that either directly or indirectly, TonB promotes loop dynamics that force bound ligands into the underlying pore. But this action of TonB does not occur by its binding to the "TonB-box" or another site within the N-domain of siderophore receptors, because neither Fhubeta nor Fepbeta contain these regions. Rather, we suggest that, in the native receptors the TonB-dependent dislodgement of the bound solute, driven by an inward contraction of the surface loops, concomitantly ejects the N-domain and releases iron into the now open channel to the periplasm. Such proposed movement of the N terminus in and out of the channel superficially reprises one of the first postulates of membrane protein function, the "ball and chain" hypothesis (74). The possibility of hydrogen bonds between the residues on the surfaces of the globular domain and the beta -barrel walls suggests an energetic barrier to this reaction mechanism, perhaps explaining the energy dependence of iron transport across the OM. For FhuA and FepA such movement of the globular domain does not cause or regulate ligand uptake but rather occurs in response to ligand internalization. This model also rationalizes the problematical uptake of siderophore antibiotics, which often double the mass of an iron complex without preventing its recognition and transport. A large, nonspecific uptake pathway, comparable to those that exist in the open beta -barrels of FepA and FhuA, is necessary to accommodate the passage of such Trojan horse antibiotics through the OM.

    ACKNOWLEDGEMENTS

We thank Ines Chen, Paul Cook, Charles Earhart, Giovanna Ferro-Luzzi Ames, and Emil Gotschlich for reading the manuscript, T. Hashimoto-Gotoh for pHSG575, and Marjorie Montague for technical assistance.

    FOOTNOTES

* This work was supported by National Science Foundation Grant MCB9709418 and National Institutes of Health Grant GM53836.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Tel.: 405-325-4969; Fax: 405-325-6111; E-mail: peklebba@ou.edu.

Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011282200

2 J. A. Coulton, personal communication.

    ABBREVIATIONS

The abbreviations used are: OM, outer membrane; LGP, ligand-gated porin; FeEnt, ferric enterobactin; Fc, ferrichrome; mAb, monoclonal antibody; LDS, lithium dodecyl sulfate; MOPS, 4-morpholinepropanesulfonic acid; PCR, polymerase chain reaction; TBS, tris-buffered saline; SulfoEGS, ethylene glycobis(sulfosuccimidylsuccinate); ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; ESR, electron spin resonance.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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