Substrate-induced transmembrane signaling in the cobalamin transporter BtuB

The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients. In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle. A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed. We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)). In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate. Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB. We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium.     

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

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

Transport-defective mutations alter the conformation of the energy-coupling motif of an outer membrane transporter.

The bacterial outer membrane transporter for vitamin B(12), BtuB, derives its energy for transport by interacting with the trans-periplasmic membrane protein TonB. This interaction with TonB occurs in part through an N-terminal segment in the BtuB sequence called the Ton box. In the present study, site-directed spin labeling of intact outer membrane preparations was used to investigate the conformation of the Ton box in wild-type BtuB and in two transport-defective mutants, L8P and V10P. In the wild-type protein, the Ton box is folded into the barrel of the transporter. The conformation of this segment is dramatically different in the transport-defective mutants L8P and V10P, where the Ton box is found to be flexible, and undocked from the transporter barrel with a greater exposure to the periplasm. In the wild-type protein, vitamin B(12) induces an undocking of the Ton box, but its addition to these transport defective mutants produces little or no change in the conformation of the Ton box. Proline substitutions at positions that do not alter transport do not alter the wild-type conformation of the Ton box; thus, the effect of substituting proline at positions 8 and 10 on the docked state of the Ton box appears to be unique. The failure of these mutants to execute the B(12) transport cycle may be a result of the altered conformation of the Ton box.     

Substrate-induced exposure of an energy-coupling motif of a membrane transporter

BtuB is an outer membrane protein responsible for the uptake of vitamin B12 by Escherichia coli. It belongs to a family of bacterial transport proteins that derive energy for transport by coupling to the trans-periplasmic energy-coupling protein TonB. Using site-directed spin labeling and EPR we investigated the structure and substrate-induced changes in the TonB box, a highly conserved region in all TonB dependent transporters that may couple to TonB. In the absence of substrate, the line widths and collision parameters from EPR are consistent with this domain existing in a structured helical conformation that contacts the barrel of the transporter. Addition of substrate converts this segment into an extended structure that is highly dynamic, disordered and probably extended into the periplasm. This structural change demonstrates that the TonB box cycles between sequestered and accessible states in a substrate-dependent fashion. In a transport defective mutant of BtuB, this conformational cycle is disrupted and the TonB box appears to be extended even in the absence of substrate. These data suggest that the TonB box extends into the periplasm and interacts with TonB only in     

Regions of Escherichia coli TonB and FepA proteins essential for in vivo physical interactions.

The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.     

Sequence changes in the ton box region of BtuB affect its transport activities and interaction with TonB protein

Uptake of cobalamins by the transporter protein BtuB in the outer membrane of Escherichia coli requires the proton motive force and the transperiplasmic protein TonB. The Ton box sequence near the amino terminus of BtuB is conserved among all TonB-dependent transporters and is the only known site of mutations that confer a transport-defective phenotype which can be suppressed by certain substitutions at residue 160 in TonB. The crystallographic structures of the TonB-dependent transporter FhuA revealed that the region near the Ton box, which itself was not resolved, is exposed to the periplasmic space and undergoes an extensive shift in position upon binding of substrate. Site-directed disulfide bonding in intact cells has been used to show that the Ton box of BtuB and residues around position 160 of TonB approach each other in a highly oriented and specific manner to form BtuB-TonB heterodimers that are stimulated by the presence of transport substrate. Here, replacement of Ton box residues with proline or cysteine revealed that residue side chain recognition is not important for function, although replacement with proline at four of the seven Ton box positions impaired cobalamin transport. The defect in cobalamin utilization resulting from the L8P substitution was suppressed by cysteine substitutions in adjacent residues in BtuB or in TonB. This suppression did not restore active transport of cobalamins but may allow each transporter to function at most once. The uncoupled proline substitutions in BtuB markedly affected the pattern of disulfide bonding to TonB, both increasing the extent of cross-linking and shifting the pairs of residues that can be joined. Cross-linking of BtuB and TonB in the presence of the BtuB V10P substitution became independent of the presence of substrate, indicating an additional distortion of the exposure of the Ton box in the periplasmic space. TonB action thus requires a specific orientation for functional contact with the Ton box, and changes in the conformation of this region block transport by preventing substrate release and repeated transport cycles.     

The Escherichia coli outer membrane cobalamin transporter BtuB: structural analysis of calcium and substrate binding, and identification of orthologous transporters by sequence/structure conservation

Gram-negative bacteria possess specialized active transport systems that function to transport organometallic cofactors or carriers, such as cobalamins, siderophores, and porphyrins, across their outer membranes. The primary components of each transport system are an outer membrane transporter and the energy-coupling protein TonB. In Escherichiacoli, the TonB-dependent outer membrane transporter BtuB carries out active transport of cobalamin (Cbl) substrates across its outer membrane. Cobalamins bind to BtuB with nanomolar affinity. Previous studies implicated calcium in high-affinity binding of cyanocobalamin (CN-Cbl) to BtuB. We previously solved four structures of BtuB or BtuB complexes: an apo-structure of a methionine-substitution mutant (used to obtain experimental phases by selenomethionine single-wavelength anomalous diffraction studies); an apo-structure of wild-type BtuB; a binary complex of calcium and wild-type BtuB; and a ternary complex of calcium, CN-Cbl and wild-type BtuB. We present an analysis of the binding of calcium in the binary and ternary complexes, and show that calcium coordination changes upon substrate binding. High-affinity CN-Cbl binding and calcium coordination are coupled. We also analyze the binding mode of CN-Cbl to BtuB, and compare and contrast this binding to that observed in other proteins that bind Cbl. BtuB binds CN-Cbl in a manner very different from Cbl-utilizing enzymes and the periplasmic Cbl binding protein BtuF. Homology searches of bacterial genomes, structural annotation based on the presence of conserved Cbl-binding residues identified by analysis of our BtuB structure, and detection of homologs of the periplasmic Cbl-binding binding protein BtuF enable identification of putative BtuB orthologs in enteric and non-enteric bacterial species.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

From the periplasmic signaling domain to the extracellular face of an outer membrane signal transducer of Pseudomonas aeruginosa: crystal structure of the ferric pyoverdine outer membrane receptor

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

Modulation by substrates of the interaction between the HasR outer membrane receptor and its specific TonB-like protein, HasB

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

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

Uptake of siderophores and vitamin B(12) through the outer membrane of Escherichia coli is effected by an active transport system consisting of several outer membrane receptors and a protein complex of the inner membrane. 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 B(12.) The active transport across the outer membrane is driven by the chemiosmotic gradient of the inner membrane and is mediated by the TonB protein. The receptor-binding domain of TonB appears to be formed by a highly conserved C-terminal amino acid sequence of approximately 100 residues. Crystal structures of two C-terminal TonB fragments composed of 85 (TonB-85) and 77 (TonB-77) amino acid residues, respectively, 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 beta-hairpin and a C-terminal beta-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-A resolution, shows a dimer with considerably reduced intermolecular interaction compared with the other known TonB structures, in particular lacking the beta-hairpin exchange.     

TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli

The energy source for active transport of iron–siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues TolQ and TolR, were absent, TonB was found associated with the outer membrane. TetA–TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.     

ExbB and ExbD do not function independently in TonB-dependent energy transduction

ExbB and ExbD proteins are part of the TonB-dependent energy transduction system and are encoded by the exb operon in Escherichia coli. TonB, the energy transducer, appears to go through a cycle during energy transduction, with the absence of both ExbB and ExbD creating blocks at two points: (i) in the inability of TonB to respond to the cytoplasmic membrane proton motive force and (ii) in the conversion of TonB from a high-affinity outer membrane association to a high-affinity cytoplasmic membrane association. The recent observation that ExbB exists in 3.5-fold molar excess relative to the molarity of ExbD in E. coli suggests the possibility of two types of complexes, those containing both ExbB and ExbD and those containing only ExbB. Such distinct complexes might individually manifest one of the two activities described above. In the present study this hypothesis was tested and rejected. Specifically, both ExbB and ExbD were found to be required for TonB to conformationally respond to proton motive force. Both ExbB and ExbD were also required for association of TonB with the cytoplasmic membrane. Together, these results support an alternative model where all of the ExbB in the cell occurs in complex with all of the ExbD in the cell. Based on recently determined cellular ratios of TonB system proteins, these results suggest the existence of a cytoplasmic membrane complex that may be as large as 520 kDa.     

The effect of calcium on the conformation of cobalamin transporter BtuB

BtuB is a β‐barrel membrane protein that facilitates transport of cobalamin (vitamin B12) from the extracellular medium across the outer membrane of Escherichia coli. It is thought that binding of B12 to BtuB alters the conformation of its periplasm‐exposed N‐terminal residues (the TonB box), which enables subsequent binding of a TonB protein and leads to eventual uptake of B12 into the cytoplasm. Structural studies determined the location of the B12 binding site at the top of the BtuB's β‐barrel, surrounded by extracellular loops. However, the structure of the loops was found to depend on the method used to obtain the protein crystals, which—among other factors—differed in calcium concentration. Experimentally, calcium concentration was found to modulate the binding of the B12 substrate to BtuB. In this study, we investigate the effect of calcium ions on the conformation of the extracellular loops of BtuB and their possible role in B12 binding. Using all‐atom molecular dynamics, we simulate conformational fluctuations of several X‐ray structures of BtuB in the presence and absence of calcium ions. These simulations demonstrate that calcium ions can stabilize the conformation of loops 3–4, 5–6, and 15–16, and thereby prevent occlusion of the binding site. Furthermore, binding of calcium ions to extracellular loops of BtuB was found to enhance correlated motions in the BtuB structure, which is expected to promote signal transduction. Finally, we characterize conformation dynamics of the TonB box in different X‐ray structures and find an interesting correlation between the stability of the TonB box structure and calcium binding. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Mechanism of ferripyoverdine uptake by Pseudomonas aeruginosa outer membrane transporter FpvA: no diffusion channel formed at any time during ferrisiderophore uptake

To get access to iron, Pseudomonas aeruginosa produces the siderophore pyoverdine (PVD), composed of a fluorescent chromophore linked to an octapeptide, and its corresponding outer membrane transporter FpvA. This transporter is composed of three domains: a β-barrel inserted into the membrane, a plug that closes the channel formed by the barrel, and a signaling domain in the periplasm. The plug and the signaling domain are separated by a sequence of five residues called the TonB box, which is necessary for the interaction of FpvA with the inner membrane TonB protein. Genetic deletion of the plug domain resulted in the presence of a β-barrel in the outer membrane unable to bind and transport PVD-Fe. Expression of the soluble plug domain with the TonB box inhibited PVD-(55)Fe uptake most likely through interaction with TonB in the periplasm. A reconstituted FpvA in the bacterial outer membrane was obtained by the coexpression of separately encoded plug and β-barrel domains, each endowed with a signal sequence and a signaling domain. This resulted in polypeptide complementation after secretion across the cytoplasmic membrane. The reconstituted FpvA bound PVD-Fe with the same affinity as wild-type FpvA, indicating that the resulting transporter is correctly folded and reconstituted in the outer membrane. PVD-Fe uptake was TonB-dependent but 75% less efficient compared to wild-type FpvA. These data are consistent with a gated mechanism in which no open channel with a complete removal of the plug domain for PVD-Fe diffusion is formed in FpvA at any point during the uptake cycle.     

Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa

Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.     

The N-terminal domain of a TonB-dependent transporter undergoes a reversible stepwise denaturation

Gram-negative bacteria contain a family of outer membrane transport proteins that function in the uptake of rare nutrients, such as iron and vitamin B(12). These proteins are termed TonB-dependent because transport requires an interaction with the inner-membrane protein TonB. Using a combination of site-directed spin labeling and chemical denaturation, we examined the site-specific unfolding of regions of the Escherichia coli vitamin B(12) transporter, BtuB. The data indicate that a portion of the N-terminal region of the protein, which occupies the lumen of the BtuB barrel, denatures prior to the unfolding of the barrel and that the free energy of folding for the N-terminus is smaller than that typically seen for globular proteins. Moreover, the data indicate that the N-terminal domain does not unfold in a single event but unfolds in a series of independent steps. The unfolding of the N-terminus is reversible, and removal of denaturant restores the native fold of the protein. These data are consistent with proposed transport mechanisms that involve a transient rearrangement or unfolding of the N-terminus of the protein, and they provide evidence of a specific protein conformation that might be an intermediate accessed during transport.     

TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport†

Cells growing in aerobic environments have developed intricate strategies to overcome the scarcity of iron, an essential nutrient. In Gram-negative bacteria, high-affinity iron acquisition requires outer membrane-localized proteins that bind iron chelates at the cell surface and promote their uptake. Transport of bound chelates across the outer membrane depends upon TonB–ExbB–ExbD, a cytoplasmic membrane-localized complex that transduces energy from the proton motive force to high-affinity receptors in the outer membrane. Upon ligand binding to iron chelate receptors, conformational changes are induced, some of which are detected in the periplasm. These structural alterations signal the ligand-loaded status of the receptor and, therefore, the requirement for TonB-dependent energy transduction. Thus, TonB interacts preferentially and directly with ligand-loaded receptors. Such a mechanism ensures the productive use of cellular energy to drive active transport at the outer membrane.     

TonB-dependent outer membrane transport: going for Baroque?

The import of essential organometallic micronutrients (such as iron-siderophores and vitamin B(12)) across the outer membrane of Gram-negative bacteria proceeds via TonB-dependent outer membrane transporters (TBDTs). The TBDT couples to the TonB protein, which is part of a multiprotein complex in the plasma (inner) membrane. Five crystal structures of TBDTs illustrate clearly the architecture of the protein in energy-independent substrate-free and substrate-bound states. In each of the TBDT structures, an N-terminal hatch (or plug or cork) domain occludes the lumen of a 22-stranded beta barrel. The manner by which substrate passes through the transporter (the "hatch-barrel problem") is currently unknown. Solution NMR and X-ray crystallographic structures of various TonB domains indicate a striking structural plasticity of this protein. Thermodynamic, biochemical and bacteriological studies of TonB and TBDTs indicate further that existing structures do not yet capture critical energy-dependent and in vivo conformations of the transport cycle. The reconciliation of structural and non-structural experimental data, and the unambiguous experimental elucidation of a detailed molecular mechanism of transport are current challenges for this field.