The TonB system actively transports large, scarce, and important nutrients through outer membrane (OM) transporters of Gram-negative bacteria using the proton gradient of the cytoplasmic membrane (CM). In Escherichia coli, the CM proteins ExbB and ExbD harness and transfer proton motive force energy to the CM protein TonB, which spans the periplasmic space and cyclically binds OM transporters. TonB has two activity domains: the amino-terminal transmembrane domain with residue H20 and the periplasmic carboxy terminus, through which it binds to OM transporters. TonB is inactivated by all substitutions at residue H20 except H20N. Here, we show that while TonB trapped as a homodimer through its amino-terminal domain retained full activity, trapping TonB through its carboxy terminus inactivated it by preventing conformational changes needed for interaction with OM transporters. Surprisingly, inactive TonB H20A had little effect on homodimerization through the amino terminus and instead decreased TonB carboxy-terminal homodimer formation prior to reinitiation of an energy transduction cycle. That result suggested that the TonB carboxy terminus ultimately interacts with OM transporters as a monomer. Our findings also suggested the existence of a separate equimolar pool of ExbD homodimers that are not in contact with TonB. A model is proposed where 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. IMPORTANCECanonical mechanisms of active transport across cytoplasmic membranes employ ion gradients or hydrolysis of ATP for energy. Gram-negative bacterial outer membranes lack these resources. The TonB system embodies a novel means of active transport across the outer membrane for nutrients that are too large, too scarce, or too important for diffusion-limited transport. A proton gradient across the cytoplasmic membrane is converted by a multiprotein complex into mechanical energy that drives high-affinity active transport across the outer membrane. This system is also of interest since one of its uses in pathogenic bacteria is for competition with the host for the essential element iron. Understanding the mechanism of the TonB system will allow design of antibiotics targeting iron acquisition.T he ability to acquire iron is the basis of a tug-of-war between host and bacterial pathogen (1, 2). Successful pathogens can acquire iron from their hosts, and in the case of Gram-negative bacteria, that is usually mediated by the TonB system, thus making the understanding of its mechanism an imperative goal (3, 4) and an attractive target for antibiotic development (5).In broad terms, the TonB system harnesses the proton motive force (PMF) of the cytoplasmic membrane (CM) to energize active transport across a ...

The TonB system energizes transport of nutrients across the outer membrane of Escherichia coli using cytoplasmic membrane proton motive force (PMF) for energy. Integral cytoplasmic membrane proteins ExbB and ExbD appear to harvest PMF and transduce it to TonB. The carboxy terminus of TonB then physically interacts with outer membrane transporters to allow translocation of ligands into the periplasmic space. The structure of the TonB carboxy terminus (residues ~150 to 239) has been solved several times with similar results. Our previous results hinted that in vitro structures might not mimic the dimeric conformations that characterize TonB in vivo. To test structural predictions and to identify irreplaceable residues, the entire carboxy terminus of TonB was scanned with Cys substitutions. TonB I232C and N233C, predicted to efficiently form disulfide-linked dimers in the crystal structures, did not do so. In contrast, Cys substitutions positioned at large distances from one another in the crystal structures efficiently formed dimers. Cys scanning identified seven functionally important residues. However, no single residue was irreplaceable. The phenotypes conferred by changes of the seven residues depended on both the specific assay used and the residue substituted. All seven residues were synergistic with one another. The buried nature of the residues in the structures was also inconsistent with these properties. Taken together, these results indicate that the solved dimeric crystal structures of TonB do not exist. The most likely explanation for the aberrant structures is that they were obtained in the absence of the TonB transmembrane domain, ExbB, ExbD, and/or the PMF.

The cytoplasmic membrane protein TonB couples the protonmotive force of the cytoplasmic membrane to active transport across the outer membrane of Escherichia coli. The uncleaved amino-terminal signal anchor transmembrane domain (TMD; residues 12 to 32) of TonB and the integral cytoplasmic membrane proteins ExbB and ExbD are essential to this process, with important interactions occurring among the several TMDs of all three proteins. Here, we show that, of all the residues in the TonB TMD, only His 20 is essential for TonB activity. When alanyl residues replaced all TMD residues except Ser 16 and His 20 , the resultant "all-Ala Ser 16 His 20 " TMD TonB retained 90% of wild-type iron transport activity. Ser 16 Ala in the context of a wild-type TonB TMD was fully active. In contrast, His 20 Ala in the wild-type TMD was entirely inactive. In more mechanistically informative assays, the all-Ala Ser 16 His 20 TMD TonB unexpectedly failed to support formation of disulfidelinked dimers by TonB derivatives bearing Cys substitutions for the aromatic residues in the carboxy terminus. We hypothesize that, because ExbB/D apparently cannot efficiently down-regulate conformational changes at the TonB carboxy terminus through the all-Ala Ser 16 His 20 TMD, the TonB carboxy terminus might fold so rapidly that disulfide-linked dimers cannot be efficiently trapped. In formaldehyde cross-linking experiments, the all-Ala Ser 16 His 20 TMD also supported large numbers of apparently nonspecific contacts with unknown proteins. The all-Ala Ser 16 His 20 TMD TonB retained its dependence on ExbB/D. Together, these results suggest that a role for ExbB/D might be to control rapid and nonspecific folding that the unregulated TonB carboxy terminus otherwise undergoes. Such a model helps to reconcile the crystal/nuclear magnetic resonance structures of the TonB carboxy terminus with conformational changes and mutant phenotypes observed at the TonB carboxy terminus in vivo.The gram-negative envelope is characterized by two concentric membranes, the inner or cytoplasmic membrane (CM) and an outer, asymmetric lipopolysaccharide and phospholipid leaflet known as the outer membrane (OM). This OM retards the passage of various hydrophobic toxins and yet allows diffusion of hydrophilic nutrients through aqueous channels provided by porin proteins. While most of the metabolic demands of gram-negative bacteria can be met by diffusible nutrients, iron demand presents a complication. In neutral oxidizing environments, bioavailable iron is limiting. To obtain it, most microbes rely upon the synthesis and secretion of high-affinity iron chelators called siderophores to capture ferric ions. Because the resultant iron-siderophore complexes exceed the diffusion limit of the OM, efficient iron retrieval depends on transporters with subnanomolar affinities for the siderophores.Release (transport) of the tightly bound ligand into the periplasmic space requires energy. Because envelope physiology and architecture preclude energy production at the OM, these transp...

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The active transport of iron siderophores and vitamin B 12 across the outer membrane (OM) of Escherichia coli requires OM transporters and the potential energy of the cytoplasmic membrane (CM) proton gradient and CM proteins TonB, ExbB, and ExbD. A region at the amino terminus of the transporter, called the TonB box, directly interacts with TonB Q160 region residues. R158 and R166 in the TonB Q160 region were proposed to play important roles in cocrystal structures of the TonB carboxy terminus with OM transporters BtuB and FhuA. In contrast to predictions based on the crystal structures, none of the single, double, or triple alanyl substitutions at arginyl residues significantly decreased TonB activity. Even the quadruple R154A R158A R166A R171A mutant TonB still retained 30% of wild-type activity. Up to five residues centered on TonB Q160 could be deleted without inactivating TonB or preventing its association with the OM. TonB mutant proteins with nested deletions of 7, 9, or 11 residues centered on TonB Q160 were inactive and appeared never to have associated with the OM. Because the 7-residuedeletion mutant protein (TonB⌬7, lacking residues S157 to Y163) could still form disulfide-linked dimers when combined with W213C or F202C in the TonB carboxy terminus, the TonB⌬7 deletion did not prevent necessary energy-dependent conformational changes that occur in the CM. Thus, it appeared that initial contact with the OM is made through TonB residues S157 to Y163. It is hypothesized that the TonB Q160 region may be part of a large disordered region required to span the periplasm and contact an OM transporter.The gram-negative cell envelope consists of an energized cytoplasmic membrane (CM) and a concentric, unenergized outer membrane (OM), separated by the aqueous periplasmic space. The OM is a diffusion barrier, protecting the cell from hydrophobic drugs, detergents, and degradative enzymes present in the environment while allowing the passive diffusion of nutrients smaller than 600 Da through the porins (56). The transport of Fe(III) siderophores or cobalamin across the OM requires the CM protonmotive force to be transduced into high-affinity transporters in the spatially separate OM. The TonB system in the CM (TonB, ExbB, and ExbD) plays a key role in energy transduction events at the OM (for recent reviews, see references 62, 76, and 77).TonB, ExbB, and ExbD form a complex in the CM through their transmembrane domains (5,22,28,31,33,34,48,60,67,73,74). TonB is present in the CM as a dimer (19, 68). The TonB/ExbB/ExbD ratio in the cell is 1:7:2, although it is not known if this value reflects the ratio of the three proteins in an energy transduction complex (23, 26). Unlike ExbB and ExbD, approximately one-third of the total cellular TonB is found associated with the OM following sucrose density gradient fractionation (50). Present data suggest that TonB achieves OM association through a shuttling process whereby the entire protein transits out of the CM following initial OM contact and associates entirely with t...