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Energy Transfer between Biological Membranes Volkmar Braun* Microbiology/Membranephysiology, University of Tuebingen, Tuebingen, Germany
A B S T R A C T The crystal structures of two transport proteins associated with a fragment of the TonB protein have been reported for the first time in two recent papers. TonB is implicated in transferring energy from the cytoplasmic membrane to the outer membrane of Gram-negative bacteria. The very similar structures of the protein complexes define the mode of interactions of TonB with active transport proteins.
*Corresponding author,
[email protected].
Published online July 21, 2006 10.1021/cb00256k CCC: $33.50 © 2006 by American Chemical Society
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he outer membrane (OM) of Gramnegative bacteria is ideal for studying mechanisms of substrate transport through membranes. The OM contains porins through which substrates pass by diffusion, specialized porins that recognize substrates entering cells by facilitated diffusion, and energy-coupled transporters. These membrane proteins are abundant when needed under appropriate growth conditions, and relatively large amounts of OMs can be isolated. The OM has unique properties. Its outer leaflet is mainly formed by the fatty acids bound to glycolipids exposed at the cell surface, and its inner leaflet is composed of a lipid covalently bound to a lipoprotein and phospholipids, mainly phosphatidylethanolamine. Proteins form -barrels in OMs. The high abundance of the proteins, -barrel structure, and unique lipid environment contribute to the isolation of these proteins in amounts and purity that allow protein crystals to be obtained. These crystals are of such a high quality that they diffract to a resolution permitting structures to be elucidated. In fact, several crystal structures of all three kinds of porins and transporters have been determined. The energy-coupled transporters are of particular interest because the OM does not contain an energy source. Energy is transferred from the cytoplasmic membrane (CM) into the OM. The proton motive force (PMF) of the CM drives substrate transport across the OM. Three proteins are involved in energy transfer from the CM into the OM:
TonB, with its N-terminus inserted in the CM and its C-proximal regions interacting with OM transporters; ExbB, which spans the CM three times, with most of the protein in the cytoplasm; and ExbD, arranged similarly as TonB. These three proteins are required for energy-coupled transport across the OM, but not for transport across the CM (1). Three principal questions have emerged: How does the TonB–ExbB–ExbD complex (Ton complex) respond to the PMF of the CM? How is the energy transferred from the CM into the OM? How do the transporters respond to the energy input? The crystal structures of five OM transport proteins have been determined. Four of these proteins transport Fe3⫹ siderophores, and one transports vitamin B12. Siderophores are secreted by bacteria and fungi and complex Fe3⫹, which otherwise forms a virtually insoluble hydroxide precipitate. The very low concentrations of these substrates and their large size exclude uptake by diffusion through porins at sufficient rates. The substrates bind to the transporters with Kd values in the nanomolar range. Extraction of the substrates from the medium and their concentration on the cell surface guarantee their availability in growth-promoting amounts. The crystal structures of all five proteins reveal the same basic transporter structures. The proteins are composed of 22 antiparallel -strands that form a -barrel (residues 161–714 in FhuA). The pore inside the -barrel is completely occluded by a globular domain, which has been called a cork, a www.acschemicalbiology.org
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Figure 1. Crystal structure of FhuA– TonB(158 –235)incorporated into the OM (3). The structure of the TonB–ExbB–ExbD complex is not known, and only the transmembrane portions are drawn in the CM. The structure of the TonB segment that connects the C-terminal crystal form to the transmembrane portion is not known and is depicted as a dashed line.
plug, or a hatch. The latter term is the most appropriate because it implies an active participation of the globular domain in substrate transport. The -strands are connected by rather large loops at the cell surface and short turns in the periplasm. The substrate-binding sites are formed by amino acid side chains of the loops and the hatch. The sites are located within the proteins well above the cell surface (Figure 1). The loops are flexible and move upon binding of the substrates; they partially or completely occlude the access of the binding site, depending on the transporter. This is most obvious in the FecA protein. Binding of the substrate diferric dicitrate induces movement of loop 7 by 11 Å and loop 8 by 15 Å, and closure of the substrate entry site results. The substrate can then no longer escape into the medium; it can only move into the periplasm. Substrate binding occurs independent of the Ton complex, but translocation across the OM through the pore of the -barrel requires energy mediwww.acschemicalbiology.org
ated by the Ton complex. Energy is also required for the release of the substrates tightly bound by ⬃10 amino acid side chains. The stereochemistry of the side chains must be altered to weaken binding. In addition, the hatch must move within or out of the -barrel so that a pore is formed through which the substrates can move into the periplasm. In the June 2, 2006, issue of Science, the crystal structures of periplasmic TonB fragments bound to BtuB (2) and to FhuA (3) are described. FhuA transports the Fe3⫹ siderophore ferrichrome, and BtuB transports vitamin B12. These long-awaited crystal structures provide much-needed structural information on energy-coupled transport across the OM of Gram-negative bacteria. Both structures delineate the interaction of TonB with the transporters. Of particular importance is the so-called Ton box in the transporters. The Ton box is located close to the N-terminus of the transporters and interacts with TonB (Figure 1). Previous results have demonstrated that certain single amino acid replacements in the Ton box of FhuA and BtuB inactivate the transporters. Amino acid replacements in Gln160 of TonB partially restore the activities of the FhuA and BtuB Ton box mutants. This has been taken as evidence that these two regions interact. This conclusion is supported by spontaneous in vivo formation of disulfide bridges between cysteine residues introduced in the Ton box of BtuB and FecA and cysteine residues introduced in region 160 of TonB. In the earlier FhuA crystal structure, the Ton box (residues 6–13) is not seen because it is flexible. In the new crystal structure, in which the TonB fragment (residues 33–239 were used for crystallization, but only residues 158–235 are observed) associates with FhuA, the FhuA Ton box forms a parallel interaction with the 3strand of TonB. The 3-strand is part of a three-stranded -sheet that also includes the 1- and 2-strands (Figure 2). The NMR
structure of TonB(152–239) reveals a fourth -strand, in antiparallel orientation, which is replaced by the -strand of the FhuA TonB box (3). The crystal structure of another TonB fragment, TonB(148–239), shows a fold similar to that of TonB(152–239), in which one -strand of the three-stranded -sheet of one monomer forms a fourth antiparallel -strand with the three-stranded -sheet of another monomer (4). Gln160 of TonB is not seen in the FhuA–TonB(158– 235) structure but can be oriented such that it forms a hydrogen bond with Thr12. In the BtuB–TonB(153–233) structure, Gln160 is seen and interacts with Asp6, Leu8, and Val10 of the BtuB Ton box (residues 6–12). The new crystal structures confirm the earlier results of genetic suppressor analyses and cysteine cross-linking experiments. Numerous additional interactions occur between the TonB fragments and the transporters. In both structures, the TonB fragments occupy approximately half of the periplasmic surface area of the transporters.
Figure 2. Interface between the FhuA Ton box and the TonB -sheet. The amino acids (carbon, white; nitrogen, blue; oxygen, red) of the FhuA Ton box are numbered (9 –15). The TonB -sheet consists of three -strands, 1, 2, and 3. T8 and T9 denote turn 8 and turn 9 in FhuA (3). VOL.1 NO.6 • 352–354 • 2006
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Of particular interest in terms of function is the position of the TonB ␣1-helix close to the hatch domain; this location allows the interaction of TonB Arg166 with Glu56 of the FhuA hatch. This interaction might be important for the dislocation of the hatch to open the pore. In BtuB–TonB(153–233), Arg158 of TonB interacts with Asp6 of the BtuB Ton box. Crystal structures are static and show only one form of a protein or protein complex. They do not reveal functional dynamics but may suggest experiments aimed at helping scientists understand the way proteins function. The determined structures of TonB fragments bound to BtuB and to FhuA do not immediately provide solutions to the energy transfer from the CM into the OM and to the conformational changes that must occur for substrate transport. In fact, binding of the TonB fragments to the transporters causes only small changes in the structures of the transporters, except for the fixation of the Ton box in a defined position. The crystal structures are likely to reflect an inactive form of the transporters, given that the substrates still occupy their binding sites and the hatches still close the -barrels. Dimeric crystal structures have been determined (4, 5), and dimeric full-length TonB has been found in vivo (6). Do they represent intermediary forms in the reaction cycle underlying energy-coupled transport? Is TonB permanently associated with transporters, or does it dissociate from the transporters and associate with other transporters? These questions are important, given that the OM can have several-hundred-thousand copies of transporters yet only a few-hundred copies of TonB. The transporters also fulfill receptor functions that may or may not depend on energy input via TonB. For example, FecA transports ferric citrate but is also essential for induction of transcription of the transport genes (7). FhuA binding of bacteriophages T1 and ⌽80 requires TonB, whereas binding of phage T5 is TonB-independent. Because 354
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mutants of phages T1 and ⌽80 that infect tonB mutants can be isolated, phage DNA uptake into cells does not require TonB; rather, binding to FhuA requires TonB. This was actually the first evidence of a TonBdependent conformational change in FhuA at the cell surface. In addition, sensitivity of cells to a protein toxin (colicin M) and a toxic peptide (microcin J25) requires FhuA and TonB. Whether FecA and FhuA fulfill such activities through the same structural changes is not known. Which roles the ExbB and ExbD proteins play in energy harvesting in the CM is also not known. The TonB– ExbB–ExbD complex must be isolated to determine whether the relative protein molar amounts of 1:7:2 found in cells is the stoichiometry of the complex. The complex must be reconstituted in artificial lipid membranes to determine whether it conducts protons. Such experiments should also involve artificially applied transmembrane potentials. A reconstituted system with wildtype and inactive mutant proteins also would provide access to physicochemical investigations, which are necessary to unravel the dynamics of energy harvesting and transfer. Finding conditions for solubilization and purification of the complex and its crystallization should be possible, because crystal structures of membrane– protein complexes, such as cytochrome oxidase, have been determined. Certainly, understanding energy-coupled transport across the OM is a long way off, but it will be well worth the effort. The benefits of any future effort will not be limited to the elucidation of how the Ton system works. The Ton system resembles the Tol system, which serves for the uptake of certain phages and bacterial toxins (colicins) and is involved in the assembly of the OM. The membrane-spanning TolA portion can functionally replace the membrane-spanning TonB portion. The TolR and TolQ proteins can partially substitute for the ExbD and ExbB proteins, and vice versa. These proteins also have sequence similariBRAUN
ties to flagellar motor proteins (MotA/ExbB/ TolQ and MotB/ExbD/TolR). For example, replacement of a conserved aspartate residue in the transmembrane region of ExbD, TolR, and MotB inactivates the Ton, Tol, and Mot functions (8). Figuring out how the Ton system functions will also help us understand how the Tol and Mot systems work, and vice versa. REFERENCES 1. Postle, K., and Kadner, R. J. (2003) Touch and go: tying TonB to transport, Mol. Microbiol. 49, 869–882. 2. Shultis, D. D., Purdy, M. D., Banchs, C. N., and Wiener, M. C. (2006) Outer membrane active transport: structure of the BtuB:TonB complex, Science 312, 1396–1399. 3. Pawelek, P. D., Croteau, N., Ng-Thow-Hing, C., Khursigara, C. M., Molseeva, N., Allaire, M., and Coulton, J. W. (2006) Structure of TonB in complex with FhuA, E. coli outer membrane receptor, Science 312, 1399–1402. 4. Peacock, R. S., Weljie, A. M., Howard, S. P., Price, F. D., Vogel, H. (2004) The solution structure of the C-terminal domain of TonB and interaction studies with TonB box peptides, J. Mol. Biol. 345, 1185–1197. 5. Ködding, J., Killig, F., Polzer, P., Howard, S. P., Diederichs, K., and Welte, W. (2005) 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, J. Biol. Chem. 280, 3022–3028. 6. Sauter, A., Howard, S. P., and Braun, V. (2003) In vivo evidence for TonB dimerization, J. Bacteriol. 185, 5742–5754. 7. Braun, V. (1995) Energy-coupled transport and signal transduction through the gram-negative outer membrane via TonB-ExbB-ExbD-dependent receptor proteins, FEMS Microbiol. Rev. 16, 295–307. 8. Braun, V., and Herrmann, C. (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.
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