Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli *

FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli . The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535–27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage (cid:1)

The cell wall of Gram-negative bacteria consists of two lipid bilayers, the outer membrane and the cytoplasmic membrane enclosing the peptidoglycan layer. A number of different transport pathways regulate the uptake of essential compounds into the cell. One class of outer membrane transporters is connected to the cytoplasmic membrane by the TonB protein; therefore, they are called TonB-dependent receptors. The three-dimensional structure of a short C-terminal fragment of TonB is available in the literature (1). One of these receptors in Escherichia coli is the ferric hydroxamate uptake system containing the integral outer membrane protein FhuA (2), which serves as a receptor for the iron siderophore ferrichrome (FC), 1 the antibiotics albomycin and rifamycin CGP 4832, colicin M, and microcin J25, and the phages T1, T5, and ⌽80. Other TonBdependent iron transporters of the outer membrane include FecA for ferric dicitrate (Cit) uptake (3), FepA for enterobactin uptake (4), and BtuB for vitamin B 12 uptake (5). The transport of all of these ligands requires energy, which is provided by the electrochemical potential of the proton gradient across the cytoplasmic membrane (proton motive force) and is mediated by the protein complexes ExbB, ExbD, and TonB (6 -8). ExbB/D is located in the cytoplasmic membrane, whereas TonB is attached to the membrane by an N-terminal hydrophobic anchor (9). The major part of TonB spans the periplasmic space to reach the outer membrane receptors.
The crystal structure of FhuA reveals a two domain architecture (10,11): a ␤-barrel consisting of 22-antiparallel strands and a globular domain at the N terminus (residues 1-160), called the cork or plug domain filling most of the interior of the barrel. Stability studies using differential scanning calorimetry experiments have shown the autonomous behavior of the cork and the ␤-barrel that unfold at different temperatures (12). The interactions between the cork domain and the ␤-barrel consist of nine salt bridges and more than 60 hydrogen bonds (11). Located at the periplasmic side of FhuA there is a short ␣-helix, the so-called "switch helix" (residues 24 -29). This ␣-helix has been found to unwind during or following ligand binding, indicating that this structural change might be a signal for TonB to bind FhuA (10,11). This unwinding was observed in the crystal structures of FhuA with bound ferrichrome (10) or albomycin (13). On the other hand, the crystal structure of FhuA with the rifamycin derivative CGP-4832 demonstrates that ligand binding causes destabilization rather than unwinding of the switch helix (14). These structures present a specific ligand binding site that is exposed to the external medium and determined by specific hydrogen bonds between the substrate and residues of both the cork and the ␤-barrel domain. The crystal structures of FepA (15), FecA (16), and BtuB (17) show similar molecular architectures. The presence of a switch helix has only been observed in the structures of FhuA and FecA but not in FepA and BtuB, implying that this structure element is not essential for TonB recognition in general. The pathway of the ligand from the binding site to the periplasm and the mechanism of its transport have not yet been elucidated. Two possibilities are discussed in the literature: 1) conformational change of the cork domain opens up a channel large enough for the siderophore to slide through (18,10) or 2) the cork domain leaves the barrel together with the bound siderophore (19).
A highly conserved motif among all TonB-dependent siderophore receptors is the TonB-box (residues 7-11: DTITV in FhuA), which plays an important role in the receptor-TonB interaction (20,21). The TonB-box is located at the periplasmic side of the cork domain close to the switch helix. Furthermore, the globular domains of FhuA and FepA are exchangeable without loss of substrate specificity. For example, a mixed mutant consisting of an FhuA-barrel and an FepA-cork retains the specificity for ferrichrome, the natural substrate for FhuA (23). Different cork-barrel combinations from several bacterial strains led to the same results (24). Complexes between wt FhuA or wt FepA with the periplasmic domain of TonB were characterized in vitro (25). However, up to now there has been no in vitro evidence for interactions between the receptor lacking the cork domain and the TonB protein, and new investigations of FepA indicated that the barrel domain alone could not behave as an active transporter (26).
The TonB protein of E. coli is composed of 239 amino acids of which 17% are proline residues. Most of these are located between residues 75 and 107, spanning the periplasmic space to link the outer membrane receptor with the cytoplasmic membrane (27). The elongated conformation of this proline-rich region has been demonstrated by NMR studies (28). This region is not essential for the process of energy transduction (29). Two other significant regions can be distinguished: 1) a hydrophobic region at the N terminus (residues 1-32) anchoring TonB to the cytoplasmic membrane. The amino acids between Ser-16 and His-20 were found to be essential for the interaction with the membrane-embedded proteins ExbB and ExbD (30) and 2) a C-terminal domain that forms the contact to the outer membrane receptor. The three-dimensional structure of a Cterminal fragment (residues 155-239) reveals a cylindershaped dimer (1). Each monomer contains three ␤-strands and a short ␣-helix arranged in a dimer so that the six ␤-strands build a large antiparallel ␤-sheet. The first 10 N-terminal amino acids of this fragment are not visible in the electron density map because of their flexibility. The structure of another energy transducing protein, TolA from Pseudomonas aeruginosa, has been solved recently (31). Despite a sequence identity of only 24% (Lalign server) the crystal structure of the periplasmic domain of TolA shows a similar structure and topology, however without dimer formation. The importance of the dimer formation for the mechanism of energy transduction is thus not yet understood. However, it has been shown that a region of TonB contributing the critical interaction with the receptor is located around amino acid 160 (32). This finding was supported by the observation that synthetic nonapeptides with sequence identity to the amino acid region between resi-dues 150 and 166 of TonB are able to inhibit the capacity of wt FhuA to transport siderophores (33).
To understand the role of the C-terminal domain of TonB in the interaction with FhuA, we have investigated FhuA-TonB interactions using purified C-terminal TonB fragments of different lengths shown in Fig. 1 (consisting of 77, 86, 96, 106, 116, or 126 amino acid residues, respectively). All TonB fragments except TonB-77 were able to form a complex with FhuA. Analytical ultracentrifugation experiments and tryptophan fluorescence measurements also showed that the fragments with 86 or more amino acid residues behave differently than TonB-77. In parallel, we analyzed the ability of these TonB fragments to inhibit ferrichrome (FC) and ferric citrate (Cit) uptake in vivo and to protect cells against attack by bacteriophage ⌽80.

EXPERIMENTAL PROCEDURES
Construction of Plasmids Encoding TonB Proteins-All constructions, with the exception of pBADTonB118, were created using PCR, and the products were first cloned into an intermediate vector (pSKII ؉ or pKSII ϩ ). The oligodeoxynucleotides used are listed in Table I. The plasmid pCSTonB30 (34), which encodes residues 33-239 of the periplasmic domain of TonB cloned into pET30a (Novagen), was used as a template to generate the four smaller tonB fragments. Standard PCR conditions were used, with US10 -US12 and US26 being the forward primers unique for each fragment as indicated, each one giving a PstI cut site on the 5Ј-end of the fragment, and US5 as the return primer, creating a HindIII restriction site on the 3Ј-end of the fragment. In combination with US5, oligonucleotide US10 was used to create pBADTB86, oligonucleotide US11 for pBADTB77, oligonucleotide US12 for pBADTB96, and oligonucleotide US26 for pBADTB106. Each fragment encodes the final number of amino acids of the periplasmic domain of TonB as specified by the TonB fragment number, i.e. pBADTB77 encodes the final 76 amino acids of the periplasmic TonB domain plus a methionine as the first amino acid. The PstI-HindIIIdigested product was then electrophoresed, and the TonB fragment isolated and cloned into PstI-HindIII-digested pBAD/gIII. The construct pBADBTB118 was obtained by digesting pMFTLP (34) with PstI and HindIII and cloning the fragment into PstI-HindIII-digested pBAD/ gIII. Each of these recombinant clones codes for an 18-amino acid (54-bp) signal sequence provided by the vector. Cloning the TonB fragment into the PstI site of pBAD/gIII downstream of this sequence also adds an 8-amino acid linker at the N-terminal side. For pTB77 to pTB126, UR134, UR135, and UR141 through UR144 were the forward FIG. 1. Amino acid sequence of the C-terminal TonB fragments used in our studies. The location of the site around residues 150 -161 known to be involved in binding to FhuA is shown in boldface (33). Structural elements derived from the crystal structure of TonB-77 are indicated (1). The amino acid sequence region predicted to form a ␤-sheet is shown underlined.
primers for each fragment as indicated, each one creating an NdeI site on the 5Ј-end of the fragment, and UR136 was the return primer, which hybridizes to the pET30a vector just downstream of the multiple cloning site and contains a Bpu1102I site. Cloning of the resulting PCR fragment back into pCSTonB30 created the plasmids pTB77-pTB126, which in each case expresses the indicated TonB fragment without a signal sequence.
Bacterial Strains, Plasmids, and Growth Conditions-The strains and plasmids used in this study are shown in Table II. The media used were Tryptone yeast extract (2xYT), nutrient broth (NB) (Difco) and Luria-Bertani media (LB). The growth temperature was 37°C for all experiments. Ampicillin was used at a concentration of 100 g/ml (Ap100). Strain AB2847⌬ara was created by P1 transduction of leu::Tn10 and ⌬ara714 from LMG194 (Invitrogen) into AB2847 (35).
Purification of FhuA-FhuA405.H 6 was expressed in E. coli strain AW 740 [⌬ompF zcb::Tn10 ⌬ompC fhuA31] (36) on plasmid pHX 405 with a his 6 tag inserted between residues 405 and 406 (37). The protein was purified as described in the literature (38) with the following changes: for binding experiments the purification was stopped before the detergent exchange from LDAO to DDAO. Fractions containing FhuA were concentrated to 10 mg/ml and dialyzed overnight against 50 mM ammonium acetate pH 8.0 with 0.05% LDAO (N,N-dimethydodecylamine-N-oxide/FLUKA).
Purification of the C-terminal TonB Fragments-C-terminal fragments of TonB (77, 86, 96, 106, 116 and 126 residues, respectively) were over-expressed in E. coli BL21(DE3) cells containing the plasmids pTB77 to pTB126 (shown in Table II) and induced at OD600 ϭ 0.7 by addition of 0.4 mM IPTG (isopropyl-␤-D-thiogalactopyranoside, Bio-Vetra). Protein expression was maintained at 37°C for 2 h. The pellets from 4 ϫ 500 ml cell culture (2xYT/Kan 50 ) were resuspended in buffer A (20 mM Tris-HCl pH 8.0/100 mM NaCl/1 mM EDTA) and the cells were broken by french press (4000 PSIG/3 passes). After centrifugation at 15,000 g for 30 min the supernatant was loaded on an SP Sepharose cation-exchange column (Amersham Biosciences) and was then washed with buffer A. TonB was eluted from the column with a NaCl gradient at a salt concentration of about 300 mM NaCl. The eluate was then desalted on a Sephadex G25 column (Amersham Biosciences) before loading onto another strong cation-exchange column (Source 15s/Amersham Biosciences). The eluted TonB protein containing about 250 mM NaCl was again desalted on a Sephadex G25 column with buffer A (no EDTA) and yielded protein at a concentration of ca. 4 mg/ml. The mobility of the fragments on 15% SDS-PAGE corresponded to their theoretical molecular masses (Fig. 2). The purification was carried out within 1 day to avoid protein degradation. For analytical ultracentrifugation and crystallization experiments an additional gel filtration step was added. The protein was concentrated up to 10 mg/ml (Amicon spin-column with YMCO 5,000) and glycerol was added to a final concentration of 10%. The TonB sample was then loaded onto a gel filtration column (Superose 12 HR 60/10, Amersham Biosciences). Binding experiments with FhuA were done with TonB fragments that were purified without this gel filtration step but mixed with 0.05% LDAO immediately before the incubation with FhuA.
Purification of the FhuA405.H 6 /TonB Complexes-Protein solutions containing FhuA (10 mg/ml) and TonB fragment (4 mg/ml), respectively, were mixed in a weight ratio of 1:2 resulting in a large molar excess of TonB in the samples. The protein mixture was then incubated overnight in the presence of 60 M ferrichrome (M r ϭ 740, Biophore Research). Glycerol was subsequently added to the protein solution to a final concentration of 10%. The sample was then applied to a Superose 12 HR 60/10 column (Amersham Biosciences), equilibrated, and eluted with the following buffer: 20 mM Tris, pH 8.0/50 mM NaCl/0.05% LDAO. The flow rate was kept at 0.1 ml/min. The protein-containing fractions were analyzed by 15% SDS-PAGE and stained with Coomassie blue (Fig. 3). For Western blots to detect TonB we used anti-TonB antiserum from rabbit as previously described (34).
Crystallization, Data Collection, and Structure Solution-The C-terminal TonB-77 fragment was crystallized under the following conditions: TonB-77 was purified as described above and concentrated to 20 mg/ml (Centricon YM 5,000). Hanging drop crystallization plates were used with 1-ml reservoir solution containing 2.0 M sodium formate and 0.1 M sodium citrate, pH 5.6, mixing 2 l of reservoir solution with 2 l of protein solution in the drop. Crystals of the size 120 ϫ 120 ϫ 120 m 3 grew at 18°C within 2 weeks (Fig. 4). For diffraction data collection single TonB-77 crystals were soaked in cryobuffer: reservoir solution with 20% glycerol for 1 min and were then flash-frozen in liquid nitrogen. X-ray diffraction data were collected at beamline ID14-4 at the Electron Synchrotron Radiation Facility in Grenoble, France. The crystals diffracted to a resolution of 2.5 Å. Raw data were processed with the program package XDS (39) to a final resolution of 2.7 Å. Higher resolution shells were omitted from the refinement process because of very high R values (Ͼ50%). The space group was determined to be P6 4 22 with the following unit cell parameters: a ϭ 61.58 Å, b ϭ 61.58 Å, c ϭ 121.95 Å, ␣ ϭ 90°, ␤ ϭ 90°, and ␥ ϭ 120°. The structure of TonB-77 was solved using molecular replacement with the program MOLREP (40) and REFMAC5 (41)  Analytical Ultracentrifugation-The purified C-terminal fragments of TonB (77, 86, 96, and 116, respectively) were analyzed by sedimentation velocity and sedimentation equilibrium experiments using an AN 60-Ti rotor 316 in a Beckman XL-A Optima equipped with an optical absorbance system (Ariel Lustig, Biozentrum Basel, Switzerland). All protein solutions were freshly purified and gel-filtrated. The buffer was 20 mM Tris, pH 8.0, and 100 mM NaCl in all experiments. Velocity sedimentation data were obtained from 0.5 mg/ml protein solutions and a rotor speed of 54,000 rpm at room temperature obtaining the sedimentation coefficient (s 20,w ). Sedimentation equilibrium experiments were done at different concentrations between 0.5 and 2 mg/ml and a rotor speed of 24,000 and 28,000 rpm at room temperature. The partial specific volume () of the proteins was calculated on the basis of the amino acid distribution (45) and was near the mean value of globular proteins 0.73 cm 3 /g. These experiments were used to determine the molecular mass (M r ), hydrodynamic radius (R H ), and the frictional ratio (f/f 0 ) (46) of the purified TonB fragments. The calculations were done with the computer program SEGAL 3 based on the numerical fitting of the sedimentation equilibrium pattern to one or two exponential functions.
Tryptophan Fluorescence of the C-terminal TonB Fragments-Fluorescence spectra were measured from TonB-77, TonB-86, TonB-96, and TonB-116, respectively, at an excitation wavelength of 295 nm over the range from 320 to 400 nm (PerkinElmer Life Sciences, L550B). The fragments were purified as described above and used at a final concentration of 0.1 mg/ml.
Assay of Bacteriophage Susceptibility-Susceptibility to bacteriophage 80i 21 was measured by dropping 5-l aliquots of 10-fold dilutions of the phage onto freshly poured overlays (100 l containing ϳ10 8 cells of the various strains added to 3 ml of LB soft agar and poured onto LB plates). The LB soft overlay, LB plates, and bacterial cultures each contained the indicated concentration of arabinose when measuring the effect of the arabinose induction level on the susceptibility of the cells to bacteriophage. The susceptibility was recorded as the Ϫlog of the highest dilution of phage that gave a confluent lysis zone of the bacterial lawn.
Assay of Siderophore-dependent Growth and Iron Transport-The ability of the strains to gain iron from either ferrichrome (FC) or ferric citrate (Cit) was assayed on NB agar plates (34). To limit the free iron CAT ATG GCA TTA AGC CGT AAT CAG CC UR135 CAT ATG CCG GCA CGA GCA CAG GCA UR136 GCT AGT TAT TGC TCA GCG G UR141 CAT ATG CCG GTT ACC AGT GTG GCT TCA UR142 CAT ATG TCA AGT ACA GCA ACG GCT GCA UR143 CAT ATG TTT GAA AAT ACG GCA CCG GCA C UR144 CAT ATG AAA CCC GTA GAG TCG CGT C available to the cells, dipyridyl was added to both the agar plates and NB soft overlay at a final concentration of 250 M. When measuring the effect of the arabinose induction level on the ability of the cells to transport iron, the various indicated levels of arabinose were added. Sterile paper discs (6-mm diameter) were saturated with 10 l of either 1 mM FC, 10 mM sodium citrate, or 100 mM sodium citrate and left to dry. The discs were then placed onto the overlays, which consisted of ϳ10 8 bacteria added to 3 ml of NB soft agar. The plates were incubated overnight at 37°C, and the diameter of rings of growth around each siderophore disc was measured (in millimeters), including the diameter of the disc. No growth was recorded as 6 mm.

RESULTS
Analysis of FhuA/TonB Interaction in Vitro-Several uptake processes across the outer membrane of Gram-negative bacteria are energized by the proton motive force of the cytoplasmic membrane. TonB from E. coli is the protein that transduces the energy from the inner membrane to the outer membrane transporter. One of the transporters is the ferric hydroxamate receptor FhuA of E. coli. The amino acid region around residue 160 of TonB is known to interact with the periplasmic side of this outer membrane (32,33). For a detailed analysis of the interaction of FhuA with TonB we prepared protein complexes of FhuA with C-terminal fragments of TonB. Gel filtration (Superose 12 column) of protein mixtures containing FhuA and the C-terminal fragment of TonB led to an elution profile with two well separated peaks, which were monitored on a 15% SDS-PAGE gel stained with Coomassie blue (Fig. 3). If a protein complex was present in the sample, the first elution peak contained both FhuA and the TonB fragment (Fig. 3, lanes B1, C1, D1, E1, and E3). The second elution peak consisted only of TonB (Fig. 3, lanes A2, B2, C2, C4, D2, E2, and E4). In the case of the shortest fragment TonB-77, the first elution peak contained only FhuA (Fig. 3, lane A1), whereas all longer fragments of TonB co-eluted with FhuA from the gel filtration column. The formation of the FhuA⅐FC⅐TonB complex was only observed in the presence of ferrichrome, which also colors the solution of the first yellowish peak (data not shown). Obviously FhuA is able to form a complex with the longer C-terminal fragments of TonB (86, 96, 106, 116, and 126). We also noticed that NaCl had to be present in the buffer at a concentration of at least 50 mM for the FhuA⅐FC⅐TonB-106 complex to be stable over a pH range from 4.6 to 8.0. To stabilize the complex we tried 10% glycerol, 1% glucose, 100 mM glycine, or 1% betaine hydrochloride, respectively. Except for betaine none of these additives led to a significant alteration in the behavior of the proteins in these binding experiments. The presence of 1% betaine in the buffer abolished complex formation between FhuA and the TonB fragments. Moreover we have shown that this purified FhuA⅐FC⅐TonB-96 complex could be disrupted by the addition of 1% betaine. In the subsequent gel filtration step with 1% betaine in the elution buffer the first peak contained FhuA alone (Fig. 3, lane C3), whereas the second peak contained TonB-96 (Fig. 3, lane C4). This FhuA fraction was colorless, whereas fractions containing FhuA with bound ferrichrome had a yellowish color, suggesting that ferrichrome  had dissociated from the receptor after the addition of betaine. It is possible that betaine replaces ferrichrome in the FhuA binding site and thereby induces the release of the TonB-fragment.
Analytical Ultracentrifugation of the C-terminal TonB Fragments-The crystal structures of TonB-88 (1) and of TonB-77 (this work) show identical dimers. This led us to investigate the aggregation state of the C-terminal TonB fragments (TonB-77, TonB-86, TonB-96, and TonB-116) in solution by analytical ultracentrifugation. Sedimentation coefficients were determined by sedimentation velocity analyses and yielded increasing values from 1.54 to 1.98 S for the fragments TonB-77 and TonB-86, respectively. The TonB fragments 96 and 116 showed significantly lower sedimentation values of 1.39 and 1.37 S, respectively. The radii calculated from the sedimentation coefficients were thus much smaller for the two longer fragments than for the two shorter ones. The frictional ratio was larger than 1.2, the typical value for spherical globular proteins, suggesting that the conformation is more elongated for these lon-ger TonB fragments. The molecular mass of the fragments was assessed from sedimentation equilibrium (see "Experimental Procedures"). Molecular masses of 8.5 and 12 kDa for the fragments TonB-96 and TonB-116, respectively, correspond very well with the theoretically calculated masses based on the amino acid sequences of these proteins being 10.8 kDa for TonB-96 and 12.8 kDa for TonB-116. The masses of the shorter fragments, TonB-77 and TonB-86, of 15 and 18.3 kDa, respectively, correspond well with the theoretical masses of the homodimers of 17.4 kDa for TonB-77 and 19.5 kDa for TonB-86. In both cases no monomeric protein were detectable in the sedimentation equilibrium profiles at 0.25, 0.5, and 1.0 mg/ml protein concentration. The results from analytical ultracentrifugation are summarized in Table III.
Trp Fluorescence of the C-terminal TonB Fragments-Another way to test the aggregation state of the TonB fragments is to measure their tryptophan fluorescence. This method is based on the fact that the molecular neighborhood of tryptophan influences its fluorescence characteristics. Each of the C-terminal TonB fragments used in this study contains only one tryptophan (Trp-213, see Fig. 1) that projects its indole group into the hydrophobic core of the TonB-77 dimer (Ref. 1 and this study). The intensity maximum of the fluorescence spectra of the two dimers TonB-77 and TonB-86 is similar at max ϭ 343 nm and at max ϭ 340 nm, respectively. The maximum for the TonB fragments 96, 106, 116, and 126 is also similar, but shifted to a shorter wavelength of max ϭ 333 nm (Table IV). We are not able to explain this blue shift, because we have no information about the environment of tryptophan 213 in the monomeric form of TonB. The fact that the fluorescence spectra of the shorter and the longer fragments is in agreement with the results of the analytical ultracentrifugation analyses indicating that the C-terminal fragments of TonB with a length of 77 and 86 amino acids, respectively, form homodimers in solution, whereas the longer fragments TonB-96 and TonB-116 remain monomeric (see Table IV).

TonB Fragments Shorter Than 96 Amino Acids Inhibit TonB Function in Vivo Very
Weakly-It has previously been demonstrated that the entire periplasmic C-terminal domain of TonB can inhibit the function of native TonB in vivo (34). Various C-terminal fragments of TonB were produced as periplasmic proteins by expression as a fusion protein with the signal sequence of FecA (34). The periplasmic C-terminal domain was shown to inhibit both ferrichrome and ferric citrate transport as well as growth on iron-limited media when iron was provided as ferrichrome or ferric citrate. In addition, induction of the periplasmic TonB fragment was shown to rescue the producing cells from the lethal effects of colicin M and bacteriophage ⌽80, both of which depend on TonB for uptake. In those studies, the smallest fragment of TonB to be assayed and shown to be inhibitory was that produced by pMFTLP, which contained the last 118 amino acids of TonB. Here, we similarly assayed fragments containing the last 77, 86, 96, and 106 amino acids of TonB, again when produced as periplasmic proteins. In this case due to the very slight inhibitions observed with some of the fragments (see Tables V and VI), we expressed the fragments as fusion proteins with the signal sequence of the GeneIII protein of the filamentous phage fd from the vector pBAD/gIII. This vector allowed higher expression than was obtained for the FecA signal sequence/TonB fusions produced by the pMalc2G vector used earlier (data not shown). As a control, we also created a GeneIII signal sequence fusion to the "LP" fragment encoding the last 118 amino acids of TonB and assayed it as well. Cells containing the plasmids were grown in varying concentrations of arabinose to induce the fusion proteins and plated on iron-deficient media containing discs soaked in ferrichrome or sodium citrate and were challenged with serial dilutions of bacteriophage ⌽80. As before, the LP fragment containing the last 118 amino acids of TonB was capable of inhibiting siderophore-dependent growth, such that at inducer concentrations of 0.02% or higher, growth on ferrichrome or ferric citrate was completely inhibited (Table V). In addition the synthesis of this fragment in the presence of 0.002% arabinose or greater was capable of rescuing the cells from the ⌽80 challenge (Table VI). As can also be seen in Tables V and VI, very similar inhibition and rescue results were observed for the C-terminal 96-and 106-amino acid TonB fragments. In contrast, there was no inhibition of siderophore-dependent growth by the C-terminal 77 amino acid TonB fragment, even when induced with 0.2% arabinose. The C-terminal 86 amino acid fragment inhibited growth only on ferrichrome and only at concentrations of 0.02% arabinose or higher (Table  V). Cells expressing the TonB-77 fragment were only poorly rescued from the ⌽80 challenge, even at the highest inducing containing ampicillin (100 g/ml) and dipyridyl (250 M) 6-mm filter discs were saturated with 10 l of 1 mM ferrichrome (Fc), 10 mM or 100 mM sodium citrate (Cit) as indicated, left to dry, and placed onto a lawn of the bacteria on media containing the indicated concentration of arabinose to induce the TonB fragments. After overnight incubation the growth zone was measured (in millimeteres) and includes the diameter of the filter disc (6 mm). Therefore a measurement of "6" indicates no visible growth around the disc. Typical results from one of three experiments are shown. Strain  a In one of the three experiments, a very faint full-sized growth ring was observed around the discs for these assay conditions. All fragments were freshly purified (see "Experimental Procedures"). The partial specific volume necessary for radius and weight determination was calculated based on the amino acid composition of the fragments. The actual molecular weight (given in kilo-Daltons) of each TonB-fragment was measured by analytical ultacentrifugation (see "Experimental Procedures") and is compared here to the theoretical molecular weight calculated by the SwissProt server (www.expasy.ch). concentration of 0.2% arabinose (Table VI)

DISCUSSION
The mechanism of energy transduction between the cytoplasmic membrane and the outer membrane via the TonB-ExbB-ExbD system is still unclear. We know, however, that the outer membrane siderophore receptors FhuA and FecA from E. coli form a complex with TonB mediating the transport of siderophores through the membrane. With respect to the crystal structure of FhuA, alone and in complex with the siderophore (10), it is assumed that the conformational changes of the receptor caused by the siderophore create a TonB binding site at the periplasmic side of the receptor. Our in vitro results correlate with this model. In the absence of the siderophore ferrichrome we observed only a very weak complex formation between FhuA and TonB, correlating with results of earlier studies (25). We also found that ferrichrome can be displaced by betaine in a purified FhuA⅐ferrichrome⅐TonB complex. This exchange of the ligand is followed by a dissociation of TonB underlining the necessity of the specific FhuA-ferrichrome in-teraction for effective binding of FhuA to TonB.
Recently it has been shown that a C-terminal fragment of TonB (TonB-86) crystallizes as a dimer (1). We were able to confirm this dimer structure by solving the structure of TonB-77 in a different crystal form. Based on these crystallographic data, it was proposed that the dimer formation of TonB is of critical importance for the mechanism of the energy transduction. Our analytical ultracentrifugation experiments support the existence of a TonB-77 dimer in solution indicating that this observation is not a crystallization artifact (Tables III  and IV). On the other hand, we found no complex formation between TonB-77 and FhuA in vitro (Fig. 3, lane A1). This observation correlates with the failure of TonB-77 to inhibit both ferrichrome uptake via FhuA and ferric citrate uptake via FecA in vivo (Table V). In addition infection of the cells by bacteriophage ⌽80 is only very weakly influenced by TonB-77 (Table VI). TonB-86 was found to be dimeric in solution as well (Table IV). This fragment, however, is able to bind to FhuA in vitro (Fig. 3, lane B1) and to interfere with ferrichrome uptake in vivo (Table V). These findings indicate the special role of the additional amino acid residues in TonB-86 compared with TonB-77 for binding to FhuA. On the other hand these residues do not influence the dimeric structure of TonB, as shown in both the crystal structure of TonB-86 and our analysis of in vitro complex formation. The addition of 10 further amino acid residues or more at the N terminus led to stable monomers in solution as we observed in case of the longer C-terminal fragments: TonB-96, TonB-106, and TonB-116 (Table IV). It was also possible to correlate these results with the in vivo inhibition studies, because each of the fragments that were monomeric in vitro strongly inhibited siderophore and bacteriophage ⌽80 uptake (Tables V and VI). Our observations agree with the results obtained for the whole periplasmic domain of TonB (residues 33-239). Sedimentation analyses showed this fragment to be monomeric in solution (25), whereas in vivo studies showed this fragment to be inhibitory for all TonB-dependent functions assayed (34). It was also shown that this fragment  binds to FhuA as a monomer (25). Sauter et al. (48) came to similar conclusions in vivo using a bacterial two-hybrid system. TonB-76 formed dimers or multimers in these experiments, whereas TonB-207 did not. Full-length TonB, containing the transmembrane part, also showed dimer formation or multimer formation. Based on the data presented here we propose that the dimer formation of the short C-terminal TonB fragments (TonB-77 and TonB-86) is an exception and not the energetically favored oligomer of native TonB. The stability of the TonB-77 dimer is supported by the formation of a 6-stranded ␤-sheet with three ␤-strands from each monomer (Fig. 6A). We suppose that a C-terminal TonB fragment longer than 96 amino acid residues can form a 4-stranded ␤-sheet by itself so that it is monomeric in solution (Fig. 6C). This hypothesis is supported by a secondary structure prediction for the longer TonB fragments, which FIG. 6. Topological diagrams derived from the structures of C-terminal TonB and TolA fragments. The arrows represent ␤-strands, and cylinders represent ␣-helices. Panel A corresponds to the structure of the C-terminal fragment TonB-86 and TonB-77 from E. coli (Ref. 1 and this study). Panel B shows the C-terminal domain of TolA from P. aeruginosa (31), which is very similar to the structure of TolA from E. coli (51). Panel C shows a putative topology for the C-terminal domain of TonB-96 derived from the structure of the TonB-77 monomer with an additional N-terminal ␤-strand consisting of 20 amino acid residues. indicates an additional zone of ␤-strand around amino acid 148 (Fig. 1). The shortest TonB fragment harboring this region is TonB-96. The additional ␤-strand might fold between ␤-strand numbers 1 and 3 (Fig. 6C). In the TonB-77 dimer this strand of the ␤-sheet is filled by the ␤-strand number 3 of a second TonB molecule (1).
A monomeric form of this proposed topology was also found in the crystal structure of TolA, a protein functionally related to TonB from the TolA⅐TolQ⅐TolR protein complex (49). The three-dimensional structure of the C-terminal domain of TolA from E. coli in complex with g3p (50) shows a very similar fold to the three-dimensional structure of the same domain of TolA from P. aeruginosa (31) despite an amino acid sequence identity of only 20%. Both TolA structures are composed of three ␤-strands and two ␣-helices in the order ␤-␤-␣-␣-␤ forming a three-stranded antiparallel ␤-sheet (Fig. 6B). A similar structure is achieved by the dimeric TonB-77 through ␤-strand swapping (31,51). The domain-swapped TonB-77 can be generated by connecting the helix from one monomer with the ␤-strand number 3 of the other monomer (Fig. 6, A and B). We hypothesize that the monomeric fragment TonB-96 folds into a three-dimensional structure similar to the domain-swapped TonB-77 dimer. Alternatively, it seems more likely that the additional 20 N-terminal amino acid residues of a TonB-96 monomer might form a ␤-strand that slides between ␤-strands 1 and 3 (Fig. 6C), building a four-stranded ␤-sheet.
A comparison of the dimerization state of the C-terminal TonB fragments and their ability to bind to FhuA (Table IV) demonstrates that the C-terminal amino acid sequence of the TonB fragments and not their aggregation state determines their binding behavior. The C-terminal fragment TonB-77, a stable dimer in solution (Tables III and IV), cannot effectively interact with the membrane receptor protein FhuA (Fig. 3,  lane A1). This finding correlates with the observation that the TonB sequence around amino acid residue 160 might contribute critical interactions to the binding of the cork domain of FhuA (21,33). This amino acid region is missing in TonB-77 (Fig. 1). On the other hand TonB-86, which forms a dimer as does TonB-77, contains the major part of the sequence around residue 160 for binding to FhuA (Fig. 1) and is able to effectively interact with the receptor molecule (Fig. 3, lane B1). In the crystal structure, these residues could not be resolved, probably due to disorder. Because the longer TonB fragments are monomeric in solution and inhibit TonB-dependent transport far more effectively than do shorter ones, we cannot find any evidence that TonB functions as a dimer in the energy transduction process.