Crystal Structure of a 92-Residue C-terminal Fragment of TonB from Escherichia coli Reveals Significant Conformational Changes Compared to Structures of Smaller TonB Fragments*
Received for publication, September 29, 2004, and in revised form, October 25, 2004 Published, JBC Papers in Press, November 2, 2004, DOI 10.1074/jbc.M411155200
Jiri Ko¨ dding‡, Frank Killig‡, Patrick Polzer‡, S. Peter Howard§, Kay Diederichs‡, and Wolfram Welte‡¶
From the‡Department of Biology, University of Konstanz, Universita¨tsstrasse 10, 78457 Konstanz, Germany and the
§Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada
Uptake of siderophores and vitamin B12 through the outer membrane ofEscherichia coliis effected by an ac- tive transport system consisting of several outer mem- brane receptors and a protein complex of the inner mem- brane. The link between these is TonB, a protein associated with the cytoplasmic membrane, which forms a large periplasmic domain capable of interacting with several outer membrane receptors,e.g.FhuA, FecA, and FepA for siderophores and BtuB for vitamin B12.The ac- tive transport across the outer membrane is driven by the chemiosmotic gradient of the inner membrane and is me- diated by the TonB protein. The receptor-binding domain of TonB appears to be formed by a highly conserved C- terminal amino acid sequence of⬃100 residues. Crystal structures of two C-terminal TonB fragments composed of 85 (TonB-85) and 77 (TonB-77) amino acid residues, re- spectively, have been previously determined (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001)J. Biol.
Chem.276, 27535–27540 and Koedding, J., Howard, S. P., Kaufmann, L., Polzer, P., Lustig, A., and Welte, W. (2004) J. Biol. Chem. 279, 9978 –9986). In both cases the TonB fragments form dimers in solution and crystallize as dimers consisting of monomers tightly engaged with one another by the exchange of a-hairpin and a C-terminal
-strand. Here we present the crystal structure of a 92- residue fragment of TonB (TonB-92), which is monomeric in solution. The structure, determined at 1.13-Å resolu- tion, shows a dimer with considerably reduced intermo- lecular interaction compared with the other known TonB structures, in particular lacking the-hairpin exchange.
The cell wall of Gram-negative bacteria consists of two lipid bilayers, the outer membrane and the cytoplasmic membrane with the peptidoglycan layer in between. A number of different transport pathways regulate the uptake of essential com- pounds into the cell. Most substances are translocated through the outer membrane by diffusion porins using a concentration gradient. However, substances occurring at very low concen-
trations like iron siderophores and vitamin B12 use specific, active, high affinity uptake systems that are driven by chemi- osmotic energy transduced to the outer membrane by the TonB protein (1). Three-dimensional structures of the following TonB- dependent receptors have been determined by x-ray crystallog- raphy: FhuA (2, 3), FepA (4), FecA (5), and BtuB (6). All of them share the same basic architecture, a 22-strand antiparallel
-barrel, which is partially filled with an N-terminal globular domain (also called plug or cork domain). The ligand binding site is exposed to the external medium, whereas the TonB binding site is located at the periplasmic side of the receptor. A peptide motif near the N terminus is conserved among all TonB-dependent receptors: D7TITV in FhuA, D12TIVV in FepA, D81ALTV in FecA, and D6TLVV in BtuB. This conserved region is called the “TonB box” (7–9). Infection ofEscherichia coli by bacteriophages T1 and 80 and uptake of bacterial toxins (Colicins M and Ia and Microcin 25) also occurs in a TonB-de- pendent manner, and TonB box motifs are found in these colicins.
Binding of the ligand to TonB-dependent receptors induces conformational changes of the cork domain. In the case of the receptor FhuA the unwinding of a short␣-helix, which directly follows the TonB box and is exposed to the periplasm (the so-called “switch-helix”), was observed (2, 3, 6). The relocation of the switch-helix likely changes the position and accessibility of the TonB box on the periplasmic side of the receptor (6, 10).
These allosteric conformational transitions, propagated from the ligand binding site to the periplasmic side, may serve to signal the ligand-loaded state of the receptor.
The TonB-dependent transporters receive their energy from the chemiosmotic gradient of the cytoplasmic membrane medi- ated by an inner membrane protein complex composed of ExbB, ExbD, and TonB (11–13). The TonB protein ofE. coliis com- posed of 239 amino acid residues and can be divided into three domains. A hydrophobic region at the N terminus (residues 1–32) anchors the TonB protein to the cytoplasmic membrane (14). Residues 12–32 are predicted to assume an␣-helical con- formation, which contains four highly conserved residues, the so-called “SHLS-motif,” which was found to be essential for the interaction with the integral membrane protein ExbB (15).
The transmembrane domain is followed by a periplasmic part with high proline content and a conserved C-terminal domain, each composed of⬃100 amino acid residues. 17% of the TonB sequence are proline residues; most of them are located be- tween residues 75 and 107 (16, 17).
Several observations indicate that the C-terminal domain of TonB (approximately residues 148 –239) interacts directly with the TonB-dependent receptors, particularly with their TonB box (18, 19). Synthetic nonapeptides corresponding to the amino acid sequence of TonB between residues 155 and 166
* This work was supported by the Deutsche Forschungsgemeinschaft and by a grant from the Natural Sciences and Engineering Research Council of Canada (to S. P. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and structure factors (code 1U07) have been deposited in the Protein Data Bank, Research Collaboratory for Struc- tural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶To whom correspondence should be addressed. Tel.: 49-7531-88- 2206; Fax: 49-7531-88-3183; E-mail: wolfram.welte@uni-konstanz.de.
T J B C Vol. 280, No. 4, Issue of January 28, pp. 3022–3028, 2005
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
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were found to be able to inhibit FhuA-dependent transport of ferrichrome in vivo (20). Complex formation between the C- terminal domain of TonB and the outer membrane receptors FhuA or FepA, respectively, has also been demonstrated by co-purification (17, 21), and disulfide cross-linking was demon- strated between cysteine substitutions in the Q160 region of TonB and the TonB box of BtuB (9) and FecA (22).
Recently the three-dimensional crystal structures of two C- terminal TonB fragments were reported. One of the fragments is composed of the C-terminal 85 amino acid residues of TonB from E. coli (“TonB-85,” residues 155–239 (23)). The other fragment is composed of the C-terminal 77 amino acid residues (“TonB-77,” residues 163–239 (21)). Both atomic models con- tain only residues 165–238, because the eight additional N- terminal residues of TonB-85 are disordered and cannot be identified in the electron density map. Apart from that the two structures are virtually identical and show two molecules tightly engaged with one another as an intertwined dimer.
Each molecule forms three-strands and a short␣-helix in the order --␣-. The dimer is stabilized by the exchange of the first two-strands, which form a-hairpin. The arrangement of the -strands in the dimer thus leads to a large 6-strand antiparallel-sheet.
The finding that the C-terminal 77 residues of TonB crystal- lized as a dimer has led to models in which a dimer of native TonB may respond to occupied high affinity receptors, possibly by rotating (23). Other recent results have indicated that the situation may be more complicated, however. In a study of C-terminal TonB fragments of increasing length (from 77 to 126 residues), it was found that fragments of 85 residues and shorter formed homodimers in solution, whereas the longer fragments were monomeric (21). In addition, the shortest frag- ment with 77 residues (which lacked glutamines 160 and 162) was unable to bind to FhuA, whereas the longer ones (which contained the glutamines) formed a complex with it as deter- mined by gel filtration studies and could inhibit the function of native TonB when producedin vivo. Furthermore, in a recent analytical ultracentrifugation and surface plasmon resonance study (24), it was found that His-tagged C-terminal fragments of 85 and 208 residues both bind to FhuA, with the shorter fragment binding as a dimer. In the absence of FhuA the longer fragment that comprises the entire periplasmic part of TonB was monomeric, whereas in the presence of FhuA there were heterotrimers composed of one receptor and two fragments.
The three-dimensional structure of the C-terminal domain of TolA that belongs to the related TolQ/R/A system was recently reported alone for thePseudomonas aeruginosaTolA (25) and in complex with the bacteriophage coat protein g3p for theE.
coliTolA (26). Despite an insignificant sequence identity (20%) between these two TolA proteins, their structures are remark- ably similar. They both crystallize as monomers and consist of a three-stranded antiparallel-sheet flanked by four␣-helices positioned on one side of the-sheet. A structure-based align- ment of the C-terminal domains of TolA from P. aeruginosa with TonB from E. coli results in an amino acid sequence identity of only 18% (25). Although TolA shares the secondary structure pattern1-2-␣-3 with TonB-77 and TonB-85, it lacks the1-2 hairpin exchange observed in the structure of TonB-77 and TonB-85 that enables the formation of a stable dimer.
Herein we report the crystal structure of a new C-terminal fragment of TonB from E. coliat 1.13-Å resolution. TonB-92 contains the C-terminal 92 amino acid residues of TonB, being only 7 residues longer than TonB-85. Its three-dimensional structure, however, differs significantly from those of TonB-85 and TonB-77, and more closely resembles that of TolA, because of the absence of the1-2 hairpin exchange. In combination
with recent results demonstrating that the longer TonB frag- ments are able to interact with FhuA more effectively and inhibit native TonB function in vivo, while the shorter frag- ments do not, these results cast new light on the question whether TonBin vivofunctions as a monomer or a dimer.
EXPERIMENTAL PROCEDURES
Protein Expression and Purification—TonB-92, a C-terminal frag- ment containing the last 92 amino acid residues of the TonB protein of E. coli, was overexpressed in BL21(DE3) cells containing the plasmid pTB92 and was subsequently purified to near homogeneity (21). Puri- fication of the SeMet1-TonB-92 was performed according to the protocol for the native TonB-92 with the following exceptions: cells were grown in M63 minimal medium (27) to anA600of 0.6, and a selected set of amino acids was then added to a medium containing lysine, phenylala- nine, and threonine at a concentration of 100 mg/liter; leucine, isoleu- cine, and valine at 50 mg/liter; and selenomethionine at 60 mg/liter. 15 Min later, 0.3 mMisopropyl 1-thio--D-galactopyranoside was added to start the overexpression of SeMet-TonB-92. Purified SeMet-TonB-92 was concentrated in a 5-kDa filter (Vivaspin), and the flow-through was dis- carded. The final yield was 1 mg of SeMet-TonB-92 per liter of culture. For crystallization, SeMet-TonB-92 was used at a concentration of 20 mg/ml.
Crystallization and Data Collection—Crystallization and data collec- tion of native TonB-92 at 1.08-Å resolution has been described else- where (28). Crystals of selenomethionine-substituted TonB-92 were grown in 100 mMimidazole, pH 8.0, 1.1Msodium citrate, and 100 mM
sodium chloride using the hanging-drop method. The crystallization drop contained 3l of protein solution and 3l of reservoir solution. For x-ray data collection the crystal was flash frozen at 100 K using reser- voir solution supplemented with 20% ethylene glycol as a cryopro- tectant. SeMet data were collected at the Swiss Light Source beamline X06SA to 2.0-Å resolution and measured at three wavelengths corre- sponding to the peak, inflection, and remote high wavelength of sele- nium. The data were processed using XDS (29). Data collection statis- tics are listed in Table I. The space group was determined to be P21; the Matthews coefficient is consistent with two molecules per asymmetric unit and a solvent content of 35%.
Structure Determination—Experimental phases were derived by the MAD method using the SeMet data. Four heavy atom sites were iden- tified by using SOLVE (30), corresponding to two methionines in the TonB-92 amino acid sequence and two molecules in the asymmetric unit. Initial protein phases were calculated by using SOLVE. These phases were further improved by solvent flattening with RESOLVE (31, 32) leading to a first polyalanine model of TonB-92 with 123 amino acid residues out of 184. The program also modeled the side chains of 50 amino acid residues. Because of the small number of heavy atoms, non-crystallographic symmetry (NCS) could not be determined from the heavy atom sites. For finding the NCS, a program2was used that identified regions with 2-fold NCS from the available main-chain frag- ments. Three corresponding C␣ atoms from each NCS region were chosen to generate a new pseudo-heavy atom coordinate file for input to RESOLVE. In the next run, RESOLVE found the 2-fold NCS in the pseudo-heavy atom sites and used it for phase improvement, leading to a new polyalanine model with 140 amino acid residues (76% of total residues). At this time 69 side chains were modeled correctly. The resulting electron density maps were taken for further manual model building using the programs O (33) and COOT (34). This model was refined against the high resolution data of the native TonB-92 using REFMAC5 (35), which is part of the CCP4 program package (36). The structure was subsequently refined with SHELXL (37). After modeling 198 water molecules and adding all hydrogen atoms, the last anisotro- pic refinement resulted in a final R-factor of 13.4% (Rfree, 18.5%).
Refinement statistics are presented in Table I. Atomic coordinates have been deposited in the Protein Data Bank with the accession code 1U07.
Dynamic Light Scattering—Dynamic light scattering experiments with purified TonB-92 were carried out using a DynaPro MS-16 – 830 instrument from Proterion Corp., High Wycombe, UK. The sample volume was 12l of protein solution at 2 mg/ml in 20 mMTris at pH 8.5 and 100 mMNaCl. Each sample was filtered through a 0.02-m pore filter (Whatman) before measurement. The time-dependent intensity signal of the scattered light was evaluated with the program Dynamics Version 6.
1The abbreviations used are: SeMet, selenomethionine; NCS, non- crystallographic symmetry.
2K. Diederichs, unpublished work..
Analytical Ultracentrifugation—Experiments were performed on a Beckman XL-A Optima analytical ultracentrifuge equipped with an AN 60-Ti rotor 316 and an optical absorbance system. All experiments were done at 20 °C with freshly prepared solution of TonB-92 at 2 mg/ml in 20 mMTris pH 8.5 containing 100 mMNaCl. The protein was finally purified by gel-permeation chromatography. In sedimentation velocity experiments absorption was scanned 86 min after the rotor reached the top speed of 52,000 rpm.
Results of sedimentation equilibrium were obtained at protein con- centrations of 0.5, 1.0, and 2 mg/ml, respectively. At a rotor speed of 34,000 rpm, equilibrium was achieved after 20 h. Data were analyzed using the software SEGAL 2.1.3 In vitro binding experiments of TonB-92 to ferrichrome-loaded FhuA were done as previously described for other C-terminal TonB-fragments (21).
RESULTS
The crystal structure of a C-terminal fragment of TonB from E. colicontaining 92 amino acid residues (“TonB-92,” residues
148 –239) was solved at 1.13-Å resolution. Because this frag- ment contains two methionine residues, we were able to gain phase information by the selenomethionine substitution method (see “Experimental Procedures”). The structure refine- ment with SHELXL resulted in a well defined model of both chains (called “a” and “b” in the following) of TonB-92 in the asymmetric unit with an R-factor of 13.4% and an Rfree of 18.5% (Fig. 1). All amino acid residues except the first two N-terminal residues of the a-chain and the first four N-termi- nal residues of the b-chain were clearly visible in the electron density map.
Description of Experimental Structure—The overall size of the molecule is ⬃50 Å by 20 Å by 20 Å. The structure of TonB-92 presents secondary structure elements in the order of␣*-1-2-␣-3 (Fig. 2). Strands1,2, and3 associate to a three-stranded -sheet. The C-terminal strand 3 is longer than1 or2 and interacts with the corresponding part of a second TonB-92 molecule by forming an intermolecular antipa- rallel -sheet. Dimerization leads to a non-crystallographic 2-fold symmetry (Fig. 1).
3See www.biozentrum.unibas.ch/personal/jseelig/AUC/software00.html.
FIG. 1.Three-dimensional structure of the dimeric TonB-92 in ribbon representation.One molecule is shown inred, and the other one isblue. The aromatic residues forming four aromatic clusters are shown inball-and-stick representation. The C-terminal-strand forms an antiparallel-sheet with the other TonB-92 molecule.
FIG. 2. Topological diagram of TonB-92 showing secondary structure elements derived from the crystal structure.-Strands are indicated byarrows, and␣-helices are indicated bycylinders. Struc- tural elements that were not observed in the structures of TonB-77 and TonB-85 are marked with anasterisk.
TABLE I
Data collection and refinement statistics of TonB-92 R-factors were calculated according to Ref. 49.
Data processing TonB-92 native TonB-92 SeMet
Peak Inflection Remote high
Wavelength (Å) 0.96862 0.97857 0.97919 0.97784
Unit cell parameters
a (Å) 22.58 22.49
b (Å) 49.32 49.16
c (Å) 72.22 71.40
b (°) 97.99 99.36
Unit cell volume (Å3) 79,600 77,900
Solvent content (%) 35.0 34.4
Resolution (Å) 10–1.08 (1.10–1.08)a 5.9–2.0 (2.1–2.0) 5.9–2.0 (2.1–2.0) 5.8–2.0 (2.1–2.0)
No. of observed reflections 426,417 (4768) 42,065 (5668) 37,950 (5121) 38,020 (5138)
No. of unique reflections 66,106 (2763) 20,072 (2878) 19,945 (2827) 20,027 (2853)
Completeness (%) 98.7 (78.5) 96.9 (86.1) 96.5 (85.1) 96.2 (85.1)
Rmeas(%) 7.9 (68.1) 5.6 (17.0) 5.6 (16.9) 5.5 (18.3)
Rmrgd-F(%) 6.5 (77.5) 6.3 (18.7) 6.5 (20.2) 6.8 (21.9)
I/Ib 12.7 (1.5) 13.3 (4.9) 12.7 (4.7) 12.9 (4.4)
S_norm/S_ano 1.25 (1.10) 1.12 (1.07) 1.24 (1.07)
Refinement statistics
Resolution range (Å) 10–1.13 (1.17–1.13)
R(%) 13.9 (19.9)
Rfree(%) 18.3
No. of nonhydrogen atoms 1,464
No. of residues (atoms) with two conformations 14 (110)
Residues with two conformations in molecule A Ser157; Asn159; Gln160; Gln175; Asn190; Ser195; Glu216; Ser222; Val226; Ile228 Residues with two conformations in molecule B Asn159; Arg171; Met201; Asn227
No. of solvent waters 201
Root mean square deviation of bond lengths (Å) 0.015 Root mean square deviation of bond angles (°) 2.966
aNumbers in parentheses correspond to the highest resolution shell.
bSize of anomalous signal as calculated in XDS/XSCALE.
Near the N terminus a segment of eleven residues (“ES1,”
Arg154-Pro164) containing the Gln160 region possesses back- bone conformation close to -sheet except for one residue, Leu156. A short helix (“␣*”) formed by six residues (Ala165- Leu170) follows which is part of a loop reversing the direction of the main chain. It is followed by a type I-hairpin composed of
1 (residues Gly174-Val182) and 2 (residues Asp189-Lys197).
The three residues PDG (Pro184-Asp185-Gly186) at the tip of the hairpin are a conserved motif for the Ton-B family and are a frequent sequence in three-residue-hairpins (39) which con- fer high turn stability even in peptides (40). After a further turn, helix ␣ (residues Glu203-Met210) and another segment with conformation close to -sheet (“ES2,” Trp213-Glu216), which is apposed to part of ES1 (Arg154-Arg158) follow. Surpris- ingly only four hydrogen bonds of this interaction stabilize the conformation of ES1: the main chains of Arg154, Leu156, and Ser157form hydrogen bonds with the main chain of Arg214and Glu216(Fig. 3). Nevertheless, the electron density of residues in ES1 is well defined (Fig. 4), and theirB-factors do not deviate from the average value. After2, the main-chain again changes direction and forms the long C-terminal strand 3 (Pro220- Thr236).3 protrudes out of the domain by about 8 residues and associates with an antiparallel3⬘of another molecule result- ing in an intermolecular-sheet3-3⬘.
Comparison with Crystal Structures of Tonb-77 and Tonb- 85—In the following, the crystal structure of TonB-92 will be compared with that shared by TonB-77 and TonB-85. The common structure of the latter two fragments contains the residues 165–238 and will be referred to as the “tight dimer.”
A superposition of one molecule from the crystal structure of TonB-92 and one from the tight dimer (Fig. 5) and their sec- ondary structure assignments (Fig. 6) show that the secondary structure elements1,2,␣, and3 are formed and arranged similarly in both structures. In the tight dimer, the two mole- cules are engaged by exchanging their1-2 hairpins with one another resulting in the formation of a six-stranded intermo- lecular antiparallel-sheet (see Fig. 5 of Ref. 21). In contrast, in the TonB-92 structure the1-2 hairpin does not exchange with another molecule but rather takes up the same place that is filled in the tight dimer with the1⬘-2⬘ hairpin from the other molecule. As seen in Fig. 5, the orientation of the-hair- pin of TonB-92 and of the tight dimer can be superimposed onto each other very well. The comparison shows that the TonB
chain has additional flexibility that is not obvious from the tight dimer structure and that results in two hinges before and after the1-2 hairpin. The backfolding of the-hairpin weak- ens the monomer-monomer interaction and leads to both a reduced length of 1 in TonB-92 and the formation of a new helix␣* (Fig. 6). Strand1 of the tight dimer starts with the residues Ala169, Leu170, and Arg171, whereas in case of TonB-92 these residues are part of the helix␣*. The presence of seven additional N-terminal residues of TonB-92 as compared with TonB-85 thus seems to abolish the1-2 exchange leading to a remarkably different crystal structure but retaining the basic arrangement of the secondary structure elements 1, 2, ␣, and3 (see Figs. 5 and 6). In the tight dimer of TonB-85, 10 N-terminal residues (Ala155-Pro164), previously shown to be important in interactions between TonB and the TonB box of the receptors (9), could not be modeled due to a high flexibility of this region. In the corresponding ES1 segment of the TonB-92 structure, residues 158 –164 possess dihedral angles close to-sheet but lack any hydrogen bonds of the backbone and the side chains to other parts of the molecule. Interactions via four hydrogen bonds occur only further N-terminally be- tween residues Arg154-Ser157and ES2.
Conversely, in the tight dimer the residues of helix␣* show
-like backbone conformation and are positioned close to the ES2 residues to which they form two hydrogen bonds. The interaction partners of ES2 thus shift by approximately ten residues from the N-terminal end of ES1 in the TonB-92 struc- ture to its C-terminal end. These differences are surprising, because TonB-77 and TonB-85 both contain all residues which in the TonB-92 structure form␣* and ES2 and in fact TonB-85 even contains most of the residues that form ES1, except for one residue, Arg154.
FIG. 3.Ball-and-stick representationof the amino acid resi- dues of TonB-92, which are involved in stabilizing the extended N-terminal ES1 segment by formation of hydrogen bonds.The bonds are shown as dotted green lines with the distances given in Angstroms (Å). The representation of the side-chain atoms is incomplete.
FIG. 4.Electron density map (2mFoⴚDFc) at 3(dark blue) and at 2(light blue) around amino acid residue Gln160of ES1.The side-chains of Asn159and Gln162show multiple conformations.
FIG. 5.Superposition of the three-dimensional structures of one molecule of TonB-92 (inred) with one molecule of the tight dimer (i.e.the structure of TonB-77 or TonB-85) (inblue).The
1-2 hairpin of the second molecule from the tight dimer is shown in yellow. The PDG-loop betweenl and2 containing residues Pro184, Asp185, and Gly186is indicated.
Five aromatic amino acid residues were found to be con- served in TonB of several Gram-negative bacteria, forming four clusters in the tight dimer. Point mutations of these residues lead to a reduced activity of TonB (41). In the two different crystal structures, these aromatic residues cluster in the same way, and the residues even have similar orientations (Fig. 1).
In particular the cluster composed of Phe180, Trp213, and Tyr215, respectively, can be superimposed upon the correspond- ing residues of TonB-85 with low deviation (Fig. 7). Phe180 resides on the exchangeable -hairpin. For this reason the aromatic cluster (Phe180, Trp213, and Tyr215) is formed by res- idues of both molecules of the tight dimer, whereas in the structure of TonB-92 all three residues belong to one molecule.
In the other cluster of the tight dimer, residues Phe202and Phe230interact by stacking of their aromatic side chains. In the TonB-92 structure a similar arrangement of these two residues is found but enlarged by Tyr163from ES1. The interaction of Phe202with Tyr163meets the criterion of an “edge on” interac- tion (42) and thus may contribute to stabilizing the folding back of the1-2 hairpin in the two hinges before1 and after2.
Tyr163has been shown to be critical for FecA function (22).
The structure of TonB-92 and the tight dimer share the formation of an intermolecular antiparallel-sheet3-3⬘. The residue pairing is, however, slightly different. In TonB-92 the center is shifted by one residue toward the C terminus compared with the tight dimer.
Oligomerization of TonB-92 in Solution, and Complex For- mation with FhuA—Experiments were carried out to compare oligomerization of TonB-92 and complex formation with FhuA in solution with other fragments, TonB-77, TonB-86, TonB-96, and TonB-116, which were studied by us recently (21). In contrast to the fact that TonB-92 forms a dimer in the crystal, we find that it behaves monomeric in solution as determined by dynamic light scattering and analytical ultracentrifugation.
Dynamic light scattering experiments show monodispersity, and the autocorrelation function of scattered light intensity can be fitted by assuming a globular protein in solution with a calculated molecular mass of 13 kDa (data not shown). This finding is consistent with the results of the analytical ultracen- trifugation experiments presenting an average molecular mass of 12 kDa and a sedimentation coefficient ofs20⫽1.34. The experimentally determined molecular weights thus correspond to the calculated molecular mass of 10.2 kDa for monomeric TonB-92. In addition, a complex of purified TonB-92 and FhuA loaded with ferrichrome could be isolated by size exclusion
chromatography (data not shown), providing evidence for the ability of TonB-92 to bind to its specific siderophore receptor FhuAin vitro.
DISCUSSION
TonB fromE. coliis known to be essential for the transduc- tion of chemiosmotic energy to TonB-dependent outer mem- brane receptors like FhuA and FepA. The atomic structure of the complete TonB (239 residues) is not yet known, but a rather elongated shape has been inferred from centrifugation and gel-filtration data (17, 24). Recently, crystal structures of two C-terminal fragments of TonB, TonB-77 (21) and TonB-85 (23), have been determined. Both fragments crystallized as an in- tertwined homodimer. Here we present the structure of TonB- 92, a C-terminal fragment of TonB fromE. colicontaining 92 amino acid residues.
Current knowledge of the molecular mechanism of TonB-de- pendent transport through the outer membrane is still very rudimentary. TonB transduces the energy that is needed for active transport of siderophores and vitamin B12through its cognate outer membrane receptors. The low copy number of TonB molecules compared with the number of TonB-dependent receptors (1) suggests that TonB probes many receptors and transduces energy only to ligand-loaded ones. Transport is initiated by binding of the ligand to the receptor binding site with submicromolar affinity. In the paradigmatic case of FhuA, the binding site is formed by residues in the external loops of FIG. 7. Superposition of one cluster of aromatic residues, Phe180, Trp213, and Tyr215, of the TonB structures.The residues from the structure of TonB-92 are colored inred. Residues of the tight dimer are given inblueandyellowdepending on which of the two TonB molecules they belong to.
FIG. 6. C-terminal amino acid se- quence of TonB fromE. coli.The posi- tion of the N termini of the fragments TonB-92, TonB-85, and TonB-77 are indi- cated byarrows. The secondary structure elements found in the structure of TonB-92 and the tight dimer (i.e.TonB-77 and TonB-85) are indicated byarrows(- strands) and boxes (helices). Conserved residues are shown with a black back- ground. The segments ES1 and ES2 are indicated bylines.
the-barrel and residues at the apices of three loops of the cork domain (2, 3). The binding is accompanied by a conformational transition propagated through the cork domain to the periplas- mic surface and results in the allosteric unwinding of a short
“switch” helix and a relocation of the periplasmic N terminus, likely accompanied by a relocation of the TonB box as found in the structures of unliganded BtuB and BtuB with bound vitamin B12(6, 43).
Several lines of evidence support the existence of a specific interaction between the C-terminal domain of TonB and the receptor, which is critical for TonB-dependent transport. A close apposition of the TonB box of the receptor with the C- terminal TonB region around Gln160has been inferred fromin vivo experiments usingtonB suppressor mutants (18), cross- linking data (8), disulfide formation in cysteine substitution mutants (9), and competitive binding of peptides (20).In vitro, complexes between receptors and TonB have been described that are more stable when the receptor contains bound ligand (17, 10, 21, 22, 44, 45). Because of the crystallographic evidence for an allosteric conformational change at the periplasmic sur- face of the receptor, TonB could in principle bind there without significantly altering its conformation, but an induced fit of TonB upon binding cannot be excluded.
Conflicting views exist about the oligomeric form at which TonB persists in the free form and when bound to its receptor.
Underin vivoconditions Sauteret al.(46) using ToxR fusions, found that the periplasmic part of TonB existed as a monomer, whereas the entire TonB and the C-terminal TonB-76 fragment formed dimers. Underin vitroconditions Koeddinget al.(21) with analytical ultracentrifugation experiments found that TonB-77 and TonB-85 formed dimers, whereas longer TonB fragments up to TonB-116 formed monomers. Under similar conditions Khursigaraet al.(24) found monomeric behavior of the periplasmic part of TonB and dimers for TonB-85. Also under similar conditions, Moeck and Letellier (17) report pre- liminary results indicating a 1:1 stoichiometry for complexes of the periplasmic part of TonB with FhuA, whereas Khursigara et al.(24) found that TonB-85 as well as the periplasmic part of TonB bound to FhuA at a 2:1 stoichiometry. A conclusion of Khursigaraet al.was that the periplasmic part of TonB, while forming monomers in absence of FhuA, formed dimers upon binding to FhuA that remained stable after dissociation from the receptor. Moreover, Khursigaraet al.observed with surface plasmon resonance experiments that the periplasmic TonB fragment displayed binding kinetics indicative of two binding sites, whereas TonB-85 displayed binding to FhuA only by a single binding site. The binding site of the periplasmic part of TonB, which was of higher affinity, apparently was not shared with TonB-85 and was responsive (increasing its affinity) to the presence of the FhuA ligand ferrichrome. This site, which must reside N-terminal of the C-terminal TonB domain, might be identical with the high affinity site in the proline-rich region described by Breweret al.(47).
Because the transport process is coupled to the use of chemi- osmotic energy, the transduction of that energy to the receptor must be triggered at a distinct moment that likely coincides with the recognition of a liganded receptor by TonB. Further- more, because the ligand has to be dissociated from its engage- ment with the high affinity binding site, one would expect the complex between TonB and the receptor to be rather tight.
Because no high affinity binding site for FhuA has so far been found in the C-terminal domain (21), a tight complex may be formed only transiently during energy transduction or its for- mation requires another high affinity site that is further N- terminal as,e.g., the site inferred from the results of Khursi- gara et al. (24) and Brewer et al. (47). Finally, because the
different TonB-dependent receptors show no overall conserva- tion of residues on the periplasmic surface, the interaction of TonB with the receptors probably relies rather on backbone interactions than on sequence-specific side-chain interactions.
In the following we will attempt to correlate all of the struc- tural results concerning TonB with these various biochemical and functional data.
Why Does the Presence of the Additional N-terminal Residues in TonB-92 as Compared with TonB-85 Change a Dimer to a Monomer in Solution and Cause Such a Significant Conforma- tional Difference in the Crystal Structure?—In solution TonB-85 and shorter C-terminal fragments of TonB behave as dimers, whereas TonB-92 and larger C-terminal fragments form monomers (data from this work and Ref. 21). Indeed, the loss of the 1-2 hairpin exchange significantly weakens the intermolecular interactions in TonB-92 as compared with the tight dimer. Nevertheless TonB-92 still forms a dimer in the crystal via its long3 strand indicating that under special circumstances it might dimerize in solution as well.
One of the keys to understanding the structural transition from the tight dimer of TonB-85 to the TonB-92 structure must be the seven additional residues at the N terminus of TonB-92.
The critical residue in the TonB-92 structure appears to be Arg154, which is the first residue missing in the TonB-85 frag- ment and which contributes one hydrogen bond to the ES1-ES2 interaction in the structure of TonB-92. Moreover, the other additional six N-terminal residues (Ser148-Pro153) do not inter- act with the rest of the molecular structure.
Is There a Role for the Two Conformations in the Transport Process?—Experimentally two crystal structures are observed that also show a simple way to derive one from the other. It is possible that these obviously stable structures of the tight dimer and of TonB-92 reflect two conformational states of na- tive full-length TonB independent of whether the dimer actu- ally formsin vivoor not. Given that, it could be that each state will play a role in two different, distinct moments of the transport process. Because the basic steps of that process are virtually unknown, only rather tentative hypotheses can be put forward.
Cross-linking data and experiments sensitive to molecular mass indicate that TonB binds the receptors at two different affinities, at a low affinity via a site in the C-terminal domain and at a higher affinity via a site further to the N terminus.
Among the subsequent associations, some likely are of higher affinity to dissociate the ligand from its micromolar binding site. The structure of TonB-92 seems to be a good candidate for a conformation that binds to the unliganded receptor at low affinity, because the Gln160region of ES1 is surface-exposed.
The TonB box may be suitably placed for the interaction only in the liganded receptor, which would explain the cross-linking results of BtuB and FecA with TonB (9, 22).
The association of ES1 with the TonB box could result in the dissociation of the weak interaction between ES1 and ES2 (which requires only four hydrogen bonds to be broken) and thus trigger a change to a conformation that resembles that of one molecule of the tight dimer. The 1-2 hairpin with its rather stable PDG turn could fold out (see Fig. 5) and would render the C-terminal TonB domain a reactive protein toward the receptor due to the many unsaturated hydrogen bonds.
This could be the conformation of the C-terminal TonB domain in the complexes with its receptor. The dimer structure itself might not form because of the smaller copy number of TonB compared with its receptors. Interestingly, a mutation of the Glycine in the PDG motif of the hairpin, G186D, abolishes growth on ferrichrome and confers strong resistance to colicins B and M (Table 3 in Traubet al.(48)), which supports the idea of an important role for the hairpin.
Other scenarios can be envisaged. Some of the proposed transport mechanisms involve a pulling force generated by conversion of chemiosmotic energy. That force may apply to the N terminus of TonB-92 after binding to the receptor and dis- rupt the ES1-ES2 bond. Subsequently ES1 may be pulled along ES2. Helix␣* may unwind, and part of its residues eventually become apposed and hydrogen-bonded to ES2. The unwinding of
␣* would abolish the hinge, which bends the main chain back- wards between ES1 and1, and the1-2 hairpin would fold out.
The two conformations of TonB-92 and of the tight dimer obviously are both rather stable, and their interconversion in solution may be inhibited by a rather high activation barrier.
Khursigaraet al.(24), describe dimer formation of the periplas- mic part of TonB by FhuA. We speculate that liganded FhuA may act as a catalyst for TonB dimerization due to its interac- tion with the ES1 segment. Moreover, Khursigara et al.(24) and Breweret al.(47) describe a high affinity interaction site of TonB with FhuA further N-terminal of the C-terminal domain.
After high affinity binding of one TonB molecule to FhuA the receptor may catalyze dimer formation of TonB if the latter is present at sufficiently high concentration. The result would be a heterotrimer formed by one liganded FhuA with a TonB tight dimer. After dissociation from FhuA, the TonB tight dimer would persist as observed by Khursigaraet al.This could serve as an inactivation mechanism for TonB, preventing TonB-de- pendent receptors from becoming blocked by an excess of inac- tive periplasmic TonB domains.
In the above scenarios, the folding out of the1-2 hairpin is presumed to lead either to a transient but tight FhuA-TonB complex of 1:1 stoichiometry or to the formation of the tight dimer with an adjacent TonB molecule. An idea of the mecha- nism by which the former complex may arise may be provided by two structures of the related domain from TolA. This energy- transducing protein, which belongs to the TolQ/R/A system is known to connect the cytoplasmic membrane with the outer membrane (38). The C-terminal domain of TolA has been crys- tallized as a monomer (25). A superposition of TonB-92 with the C-terminal fragment of TolA is given in Fig. 8 and shows a close superposition of the three-strands and the long␣-helix.
A second structure shows this TolA domain complexed as a monomer with a bacteriophage coat protein, with which it interacts during infection (26). The association shows an inter- molecular-sheet formed and stabilized by intermolecular hy- drogen bonds between TolA and G3p.
At present, the details of the interactions and conformational changes that take place during energy-dependent transport involving TonB and its receptors remain far from clear. How- ever, the two structures of the tight dimer and TonB-92 repre- sent constraints, which a final model of transport will have to take account of, and perhaps clues to the dynamic mechanism
by which TonB interacts with the receptors and transduces energy to the transport process.
Acknowledgments—We thank the staff at the X06SA (Swiss Light Source/Switzerland) synchrotron beamline for their support. We are grateful to Ariel Lustig from the Biozentrum Basel/Switzerland for performing the analytical ultracentrifugation of TonB-92. We also thank Ramon Kanaster for assisting us in purifying TonB-92 and Kinga Gerber for helpful discussions.
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one molecule of TonB-92 (red) and the C-terminal domain of TolA fromE. coli(blue) (pdb accession code: 1Tol, Lubkowskiet al.(26)).The N-terminal␣-helix of TolA, which is not present in TonB, is not shown.
