TCDB is operated by the Saier Lab Bioinformatics Group

2.C.1 The TonB-ExbB-ExbD/TolA-TolQ-TolR Outer Membrane Receptor Energizers and Stabilizers (TonB/TolA) Family

The TonB heterotrimeric complexes span the cytoplasmic membrane and the periplasm and interact with outer membrane receptors in Gram-negative bacteria. Homologues have been found only in Gram-negative bacteria and cyanobacteria. E. coli possesses two paralogous systems, the TonB-ExbB-ExbD system and the TolA-TolQ-TolR system. Corresponding proteins have been identified in other Gram-negative bacteria. TonB (239 aas) and TolA (412 aas) of E. coli both span the cytoplasmic membrane once near their N-termini, span the periplasm as α-helices and interact with the outer membrane. They are not demonstrably homologous, but they are believed to serve comparable functions. It has been suggested that TonB or its C-terminal domain shuttles between the cytoplasmic and outer membranes as part of the energy transduction process. ExbB (244 aas) and TolQ (230 aas) are paralogous. They span the membrane 3 times with their N-termini in the periplasm and most of the protein mass localized to the cytoplasmic side of the membrane. ExbD (141 aas) and TolR (142 aas) are also paralogous. They span the membrane near their N-termini with remaining parts of the proteins in the periplasm. The TonB system energizes transport (uptake) via OMR-type porins (TC #1.B.14) of vitamin B12, iron-siderophores, group B colicins and the DNA of filamentous bacteriophage such as φ80 and T1. It may also be involved in the extrusion of drugs and organic solvents from Pseudomonas putida (Godoy et al., 2001), but the mechanism is not known. The TolA system transports group A colicins and the DNA of other filamentous phages. Colicin import requires close proximity of the inner and outer membranes. Loss of one of the TolA-TolQ-TolR proteins results in loss of periplasmic enzymes and increased sensitivity to drugs and bile salts. Surface localization of O-antigen lipopolysaccharide in E. coli depends on the TolA protein, possibly explaining the leakiness of TolA mutants. The TolA/Pal system has also been reported to be necessary for the uptake of certain solutes (sugars, polyols, amino acids) (Llamas et al., 2003), but the mechanism was not investigated. TolR may rotate (Zhang et al. 2009).

Microcin E492 (TC #1.C.58.1.1) kills E. coli and other enterobacteria in a mechanism that depends on TonB, ExbBD, energy, and OMRs such as FepA (Destoumieux-Garzon et al., 2003). This energy-dependent system promotes import acoss the outer membrane and into the inner membrane where microcin E492 exerts its action.  Just how the energy cycle functions is still controversial, but Gresock et al. 2015 have provided evidence that the cycle involves monomer/dimer transitions. They proposed a model in which interaction of TonB homodimers with ExbD homodimers initiates the energy transduction cycle, and, ultimately, the ExbD carboxy terminus modulates interactions of a monomeric TonB carboxy terminus with OM transporters. After TonB exchanges its interaction with ExbD for interaction with a transporter, ExbD homodimers undergo a separate cycle needed to re-energize them (Gresock et al. 2015).

The TolA/TolQR system interacts with other proteins that appear to play a role in transport of colicins such as Colicin A and Colicin E9 (Hands et al., 2005). These include the periplasmic TolB (430 aas; spP19935), the periplasmic YbgF (263 aas; spP45955), and the outer membrane peptidoglycan-associated protein, Pal (173 aas; spP07176). These proteins probably all interact with each other, and several interact with Colicin A (Journet et al., 2001). They probably also interact with trimeric porins and OmpA. TolB interacts with the translocation domain of TolB via the TolB box located in the N-terminal translocation domain of the enzymatic E colicins (Hands et al., 2005). Based on the NMR solution structure, the periplasmic domain of TolR forms a C 2-symmetric dimer consisting of a strongly curved eight-stranded beta-sheet, generating a large deep groove on one side, while four helices cover the other face of the sheet (Parsons et al., 2008). This domain may interact with other components of the Pal/Tol system, particularly TolQ.  Interactions between the Tol subunits have been mapped, and evidence for rotation of the TolR TMS have been presented (Zhang et al. 2009; Zhang et al. 2011).

To penetrate the target cell, colicins bind to an outer membrane receptor at the cell surface and then translocate their N-terminal domain through the outer membrane and the periplasm. Once fully translocated, the N-terminal domain triggers entry of the catalytic C-terminal domain by an unknown process. Colicin K uses the Tsx nucleoside-specific receptor for binding at the cell surface, the OmpA protein for translocation through the outer membrane, and the TolABQR proteins for the transit through the periplasm. Barneoud-Arnoulet et al. (2010) described how the colicin K N-terminal domain (KT) interacts with the components of its transit machine in the periplasm. Upon production of KT in wild-type strains, cells became partly resistant to Tol-dependent colicins and sensitive to detergent and released periplasmic proteins and outer membrane vesicles, suggesting that KT interacts with and titrates components of its import machine (Barnéoud-Arnoulet et al., 2010). KT interacts with TolA, TolB and TolR. TolQ and the colicin translocation domain also interact.

Fission of bacterial cells involves the co-ordinated invagination of the envelope layers. Invagination of the cytoplasmic membrane (IM) and peptidoglycan (PG) layer is likely driven by the septal ring organelle. Invagination of the outer membrane (OM) in Gram-negative species is thought to occur passively via its tethering to the underlying PG layer with generally distributed PG-binding OM (lipo)proteins. The Tol-Pal system is energized by the proton motive force and is well conserved in Gram-negative bacteria. It consists of five proteins that can connect the OM to both the PG and IM layers via protein-PG and protein-protein interactions. As noted above, the system is needed to maintain full OM integrity, and for class A colicins and filamentous phages to enter cells. All five components accumulate at constriction sites in Escherichia coli, and mutants lacking an intact system suffer delayed OM invagination (Gerding et al., 2006). They contain large OM blebs at constriction sites and cell poles. The Tol-Pal system apparently constitutes a dynamic subcomplex of the division apparatus in Gram-negative bacteria that consumes energy to establish transient trans-envelope connections at or near the septal ring to draw the OM onto the invaginating PG and IM layers during constriction (Gerding et al., 2007). ExbD has a periplasmic domain that is structurally similar to siderophore binding proteins (Garcia-Herrero et al., 2007).

ExbB-ExbD are homologous to (but distantly related to) the MotA-MotB proteins of the Mot family (TC# 1.A.45). The latter proteins serve as the motor of the bacterial flagellum. While the MotA-B proteins have been shown to provide a transmembrane proton translocation pathway, the same has not yet been demonstrated for the ExbB-D proteins. It is expected, however, that they will serve this function and thereby energize outer membrane transport. It is likely that all of these proteins can be considered both as proton channel proteins and as pmf-dependent energizers, and that they belong in a single family.

Braun and Herrmann (2004) have proposed that at least three well-conserved transmembrane residues in ExbB (or TolQ) comprise the proton pathway. These proposed channel residues in ExbB are Thr148 in TMS2 and glu176 and Thr181 in TMS3. The first two are strictly conserved in all ExbB and TolQ homologues, and the third is almost strictly conserved in all MotA homologues as well. Asp25 in ExbD may also comprise part of this proton pathway (Braun and Herrmann, 2004). The only protonatable residue in the TMS of TonB, a histidyl residue can be replaced by a non-protonatable Gln without loss of activity. Thus, the TonB TMS is not on a proton conductance pathway. It only indirectly responds to pmf, probably via ExbD (Swayne and Postle, 2011).

Bacteria producing endonuclease colicins are protected against the cytotoxic activity by a small immunity protein that binds with high affinity and specificity to inactivate the endonuclease. This complex is released into the extracellular medium, and the immunity protein is jettisoned upon binding of the complex to susceptible cells. At what stage during infection does immunity protein release occur? Duche et al., (2006) constructed a hybrid immunity protein composed of the enhanced green fluorescent protein (EGFP) fused to the colicin E2 immunity protein (Im2) to enhance its detection. The EGFP-Im2 protein bound the free colicin E2 with a 1:1 stoichiometry and specifically inhibited its DNase activity. The addition of this hybrid complex to susceptible cells revealed that release of the hybrid immunity protein is a time-dependent process, achieved 20 min after the addition of the complex to the cells. Complex dissociation required a functional translocon formed by the BtuB protein and one porin (either OmpF or OmpC) and a functional import machinery formed by the Tol proteins. Cell fractionation and protease susceptibility experiments indicated that the immunity protein does not cross the cell envelope during colicin import. These observations suggest that dissociation of the immunity protein occurs at the outer membrane surface and requires full translocation of the colicin E2 N-terminal domain (Duche et al., 2006).

The structure of BtuB outer membrane receptor (OMR; 1.B.14.3.1) and the FhuA OMR (1.B.14.1.4) complexed with the C-terminal domain of TonB (2.C.1.1.1), the energy transmitter to the OMR from the EBDxb energizer, shows TonB binding to the TonB box in the OMRs. TonB binding causes the TonB box to form a β-strand, forming a β-sheet with TonB's own β-strand. This is consistent with a mechanical 'pulling' mechanism of transport (Shultis et al., 2006). The conserved TonB arginine 166 is oriented to form multiple contacts with the FhuA 'cork', the globublar domain enclosed by the β-barrel (Pawelek et al., 2006).

TonB-dependent transporters bind and transport ferric chelates, vitamin B12, nickel complexes, and carbohydrates. The transport process requires energy in the form of the pmf and the TonB-ExbB-ExbD complex to transduce this energy to the outer membrane. The siderophore substrates range in complexity from simple small molecules such as citrate to large proteins such as serum transferrin and hemoglobin. Expression can be regulated by metal-dependent regulators, σ/anti-σ factors, small RNAs, and a riboswitch (Noinaj et al., 2010). Noinaj et al. (2010) summarized the regulation, structure and function of these systems.

ExbB and ExbD harness the proton gradient to energize TonB, which directly contacts and transmits this energy to ligand-loaded transporters. In E. coli, the periplasmic domain of ExbD appears to transition from proton motive force-independent to proton motive force-dependent interactions with TonB, catalyzing the conformational changes of TonB. While all regions except the extreme amino terminus of ExbD are indispensable for function, distinct roles for the amino and carboxy terminal regions of the ExbD periplasmic domain have been determined (Ollis et al., 2012). Like residue D25 in the ExbD transmembrane domain, periplasmic residues 42-61 facilitate the conformational response of ExbD to proton motive force. This region appears important for transmitting signals between the ExbD transmembrane domain and carboxy terminus. The carboxy terminus, encompassing periplasmic residues 62-141, are required for initial assembly with the periplasmic domain of TonB, a stage of interaction required for ExbD to transmit its conformational response to proton motive force to TonB. Residues 92-121 are important for all three interactions; ExbD homodimers, TonB-ExbD heterodimers, and ExbD-ExbB heterodimers. The distinct requirement of this ExbD region for interaction with ExbB raised the possibility of direct interaction with the few residues of ExbB that occupy the periplasm. 

The TonB-ExbB-ExbD motor harnesses the pmf across the bacterial inner membrane to couple energy to transporters in the outer membrane, facilitating uptake of nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Celia et al. 2016 presented the first crystal structure of ExbB-ExbD and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. They subsequently used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer, with implications for motor function (Celia et al. 2019).

The generalized transport process energized by TonB-type systems is:

substrate (out) substrate (periplasm).

References associated with 2.C.1 family:

Barnéoud-Arnoulet, A., M. Gavioli, R. Lloubès, and E. Cascales. (2010). Interaction of the colicin K bactericidal toxin with components of its import machinery in the periplasm of Escherichia coli. J. Bacteriol. 192: 5934-5942. 20870776
Bell, P.E., C.T. Nau, J.T. Brown, J. Konisky, and R.J. Kadner. (1990). Genetic suppression demonstrates interaction of TonB protein with outer membrane transport proteins in Escherichia coli. J. Bacteriol. 172: 3826-3829. 2193917
Benevides-Matos, N., C. Wandersman, and F. Biville. (2008). HasB, the Serratia marcescens TonB paralog, is specific to HasR. J. Bacteriol. 190(1):21-7. 17951376
Braun, V. and C. Herrmann. (2004). Point mutations in transmembrane helices 2 and 3 of ExbB and TolQ affect their activities in Escherichia coli K-12. J. Bacteriol. 186: 4402-4406. 15205446
Braun, V., H. Pilsl, and P. Gross. (1994). Colicins: structures, modes of action, transfer through membranes and evolution. Arch. Microbiol. 161: 199-206. 8161282
Calvopiña, K., P. Dulyayangkul, K.J. Heesom, and M.B. Avison. (2019). TonB-dependent uptake of β-lactam antibiotics in the opportunistic human pathogen Stenotrophomonas maltophilia. Mol. Microbiol. [Epub: Ahead of Print] 31773806
Celia, H., I. Botos, X. Ni, T. Fox, N. De Val, R. Lloubes, J. Jiang, and S.K. Buchanan. (2019). Cryo-EM structure of the bacterial Ton motor subcomplex ExbB-ExbD provides information on structure and stoichiometry. Commun Biol 2: 358. 31602407
Celia, H., N. Noinaj, S.D. Zakharov, E. Bordignon, I. Botos, M. Santamaria, T.J. Barnard, W.A. Cramer, R. Lloubes, and S.K. Buchanan. (2016). Structural insight into the role of the Ton complex in energy transduction. Nature 538: 60-65. 27654919
Destoumieux-Garzón, D., X. Thomas, M. Santamaria, C. Goulard, M. Barthélémy, B. Boscher, Y. Bessin, G. Molle, A.-M. Pons, L. Letellier, J. Peduzzi, and S. Rebuffat. (2003). Microcin E492 antibacterial activity: evidence for a TonB-dependent inner membrane permeabilization on Escherichia coli. Mol. Microbiol. 49: 1031-1041. 12890026
Duche D., A. Frenkian, V. Prima, R. Lloubes. (2006). Release of immunity protein requires functional endonuclease colicin import machinery. J Bacteriol. 188: 8593-8600. 17012383
Garcia-Herrero, A., R.S. Peacock, S.P. Howard, and H.J. Vogel. (2007). The solution structure of the periplasmic domain of the TonB system ExbD protein reveals structural homology with siderophore-binding proteins. Mol. Microbiol. 66(4): 872-889. 17927700
Gaspar, J.A., J.A. Thomas, C.L. Marolda, and M.A. Valvano. (2000). Surface expression of O-specific lipopolysaccharide in Escherichia coli requires the funtion of the TolA protein. Molec. Microbiol. 38: 262-275. 11069653
Gerding, M.A., Y. Ogata, N.D. Pecora, H. Niki, and P.A. de Boer PA. (2007). The trans-envelope Tol-Pal complex is part of the cell division machinery and required for proper outer-membrane invagination during cell constriction in E. coli. Mol. Microbiol. 63: 1008-1025. 17233825
Godoy, P., M.I. Ramos-González, and J.L. Ramos. (2001). Involvement of the TonB system in tolerance to solvents and drugs in Pseudomonas putida DOT-T1E. J. Bacteriol. 183: 5285-5292. 11514511
Gouaux, E. (1997). The long and short of colicin action: the molecular basis for the biological activity of channel-forming colicins. Structure 5: 313-317. 9083116
Gresock MG., Kastead KA. and Postle K. (2015). From Homodimer to Heterodimer and Back: Elucidating the TonB Energy Transduction Cycle. J Bacteriol. 197(21):3433-45. 26283773
Hands, S.L., Holland, L.E., Vankemmelbeke, M., Fraser, L., Macdonald, C.J., Moore, G.R., James, R., and Penfold, C.N. (2005). Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box. J Bacteriol. 187: 6733-6741. 16166536
Hernández-Montalvo, V., F. Valle, F. Bolivar, and G. Gosset. (2001). Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system. Appl. Microbiol. Biotechnol. 57: 186-191. 11693918
Journet, L., E. Bouveret, A. Rigal, R. Lloubes, C Lazdunski, and H. Bénédetti. (2001). Import of colicins across the outer membrane of Escherichia coli involves multiple protein interactions in the periplasm. Mol. Microbiol. 42: 331-344. 11703658
Kadner, R.J. (1990). Vitamin B12 transport in Escherichia coli: energy coupling between membranes. Mol. Microbiol. 4: 2027-2033. 2089218
Klebba, P.E. (2016). ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition. J. Bacteriol. 198: 1013-1021. 26787763
Lazdunski, C., E. Bouveret, A. Rigal, L. Journet, R. Lloubès, and H. Bénédetti. (2000). Colicin import into Escherichia coli cells requires the proximity of the inner and outer membranes and other factors. Int. J. Med. Microbiol. 290: 337-344. 11111908
Lazzaroni, J.C., P. Germon, M.-C. Ray, and A. Vianney. (1999). The Tol proteins of Escherichia coli and their involvement in the uptake of biomolecules and outer membrane stability. FEMS Microbiol. Lett. 177: 191-197. 10474183
Letain, T.E. and K. Postle. (1997). TonB protein appears to transduce energy by shuttling between the cytoplasmic membrane and the outer membrane in Escherichia coli. Mol. Microbiol. 24: 271-283. 9159515
Llamas, M.A., J.J. Rodríguez-Herva, R.E.W. Hancock, W. Bitter, J. Tommassen, and J.L. Ramos. (2003). Role of Pseudomonas putida tol-oprL gene products in uptake of solutes through the cytoplasmic membrane. J. Bacteriol. 185: 4707-4716. 12896989
Muller, M.M., A. Vianney, J.-C. Lazzaroni, R.E. Webster, and R. Portalier. (1993). Membrane topology of the Escherichia coli TolR protein required for cell envelope intergity. J. Bacteriol. 175: 6059-6061. 8376353
Noinaj, N., M. Guillier, T.J. Barnard, and S.K. Buchanan. (2010). TonB-dependent transporters: regulation, structure, and function. Annu. Rev. Microbiol. 64: 43-60. 20420522
Ollis AA., Kumar A. and Postle K. (2012). The ExbD periplasmic domain contains distinct functional regions for two stages in TonB energization. J Bacteriol. 194(12):3069-77. 22493019
Parsons, L.M., A. Grishaev, and A. Bax. (2008). The periplasmic domain of TolR from Haemophilus influenzae forms a dimer with a large hydrophobic groove: NMR solution structure and comparison to SAXS data. Biochemistry 47: 3131-42. 18269247
Pawelek, P.D., N. Croteau, C. Ng-Thow-Hing, C.M. Khursigara, N. Moiseeva, M. Allaire, and J.W. Coulton. (2006). Structure of TonB in complex with FhuA, E. coli outer membrane receptor. Science 312: 1399-1402. 16741125
Postle, K. (1993). TonB protein and energy transduction between membranes. J. Bioenerg. Biomembr. 25: 591-601. 8144488
Postle, K. and R.J. Kadner. (2003). Touch and go: tying TonB to transport. Mol. Microbiol. 49: 869-882. 12890014
Qiu, G.W., W.J. Lou, C.Y. Sun, N. Yang, Z.K. Li, D.L. Li, S.S. Zang, F.X. Fu, D.A. Hutchins, H.B. Jiang, and B.S. Qiu. (2018). Outer Membrane Iron Uptake Pathways in the Model Cyanobacterium Synechocystis sp. Strain PCC 6803. Appl. Environ. Microbiol. 84:. 30076192
Rassam, P., K.R. Long, R. Kaminska, D.J. Williams, G. Papadakos, C.G. Baumann, and C. Kleanthous. (2018). Intermembrane crosstalk drives inner-membrane protein organization in Escherichia coli. Nat Commun 9: 1082. 29540681
Roof, S.K., J.D. Allard, K.P. Bertrand, and K. Postle. (1991). Analysis of Escherichia coli TonB membrane topology by use of PhoA fusions. J. Bacteriol. 173: 5554-5557. 1885532
Santos, T.M., T.Y. Lin, M. Rajendran, S.M. Anderson, and D.B. Weibel. (2014). Polar localization of Escherichia coli chemoreceptors requires an intact Tol-Pal complex. Mol. Microbiol. 92: 985-1004. 24720726
Shrivastava, R., X. Jiang, and S.S. Chng. (2017). Outer membrane lipid homeostasis via retrograde phospholipid transport in Escherichia coli. Mol. Microbiol. 106: 395-408. 28815827
Shultis, D.D., M.D. Purdy, C.N. Banchs, and M.C. Wiener. (2006). Outer membrane active transport: structure of the BtuB:TonB complex. Science 312: 1396-1399. 16741124
Smajs, D. and G.M. Weinstock. (2001). The iron- and temperature-regulated cjrBC genes of Shigella and enteroinvasive Escherichia coli strains code for colicin Js uptake. J. Bacteriol. 183: 3958-3966. 11395459
Stolz, J., H.J. Wöhrmann, and C. Vogl. (2005). Amiloride uptake and toxicity in fission yeast are caused by the pyridoxine transporter encoded by bsu1+ (car1+). Eukaryot. Cell. 4: 319-326. 15701794
Swayne, C. and K. Postle. (2011). Taking the Escherichia coli TonB transmembrane domain "offline"? Nonprotonatable Asn substitutes fully for TonB His20. J. Bacteriol. 193: 3693-3701. 21665976
Vianney, A., T.M. Lewin, W.F. Beyer, Jr., J.C. Lazzaroni, R. Portalier, and R.E. Webster. (1994). Membrane topology and mutational analysis of the TolQ protein of Escherichia coli required for the uptake of macromolecules and cell envelope integrity. J. Bacteriol. 176: 822-829. 8300535
Vogl, C., C.M. Klein, A.F. Batke, M.E. Schweingruber, and J. Stolz. (2008). Characterization of Thi9, a novel thiamine (Vitamin B1) transporter from Schizosaccharomyces pombe. J. Biol. Chem. 283: 7379-7389. 18201975
Zhai, Y.F., W. Heijne, and M.H. Saier, Jr. (2003). Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB. Biochim. Biophys. Acta 1614: 201-210. 12896813
Zhang, H.H., D.R. Blanco, M.M. Exner, E.S. Shang, C.I. Champion, M.L. Phillips, J.N. Miller, and M.A. Lovett. (1999). Renaturation of recombinant Treponema pallidum rare outer membrane protein 1 into a trimeric, hydrophobic, and porin-active conformation. J. Bacteriol. 181: 7168-7175. 10572117
Zhang, X.Y., E.L. Goemaere, N. Seddiki, H. Célia, M. Gavioli, E. Cascales, and R. Lloubes. (2011). Mapping the interactions between Escherichia coli TolQ transmembrane segments. J. Biol. Chem. 286: 11756-11764. 21285349
Zhang, X.Y., E.L. Goemaere, R. Thomé, M. Gavioli, E. Cascales, and R. Lloubès. (2009). Mapping the interactions between escherichia coli tol subunits: rotation of the TolR transmembrane helix. J. Biol. Chem. 284: 4275-4282. 19075020