Dataset Information

Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

ABSTRACT: In gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains of TonB and ExbD is required, with the conformational response of TonB to presence or absence of proton motive force being modulated through ExbD. A region (residues 92 to 121) within the ExbD periplasmic domain was previously identified as being important for TonB interaction. Here, the specific sites of periplasmic domain interactions between that region and the TonB carboxy terminus were identified by examining 270 combinations of 45 TonB and 6 ExbD individual cysteine substitutions for disulfide-linked heterodimer formation. ExbD residues A92C, K97C, and T109C interacted with multiple TonB substitutions in four regions of the TonB carboxy terminus. Two regions were on each side of the TonB residues known to interact with the TonB box of TonB-gated transporters, suggesting that ExbD positions TonB for correct interaction at that site. A third region contained a functionally important glycine residue, and the fourth region involved a highly conserved predicted amphipathic helix. Three ExbD substitutions, F103C, L115C, and T121C, were nonreactive with any TonB cysteine substitutions. ExbD D25, a candidate to be on a proton translocation pathway, was important to support efficient TonB-ExbD heterodimerization at these specific regions.

SUBMITTER: Ollis AA

PROVIDER: S-EPMC3370880 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

Ollis Anne A AA Postle Kathleen K

Journal of bacteriology 20120406 12

In gram-negative bacteria, the cytoplasmic membrane proton-motive force energizes the active transport of TonB-dependent ligands through outer membrane TonB-gated transporters. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD couple the proton-motive force to conformational changes in TonB, which are hypothesized to form the basis of energy transduction through direct contact with the transporters. While the role of ExbB is not well understood, contact between periplasmic domains ...[more]

PMID: 22493017

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Project description:In Gram-negative bacteria, the cytoplasmic membrane protein TonB transmits energy derived from proton motive force to energize transport of important nutrients through TonB-dependent transporters in the outer membrane. Each transporter consists of a beta barrel domain and a lumen-occluding cork domain containing an essential sequence called the TonB box. To date, the only identified site of transporter-TonB interaction is between the TonB box and residues ∼158 to 162 of TonB. While the mechanism of ligand transport is a mystery, a current model based on site-directed spin labeling and molecular dynamics simulations is that, following ligand binding, the otherwise-sequestered TonB box extends into the periplasm for recognition by TonB, which mediates transport by pulling or twisting the cork. In this study, we tested that hypothesis with the outer membrane transporter FepA using in vivo photo-cross-linking to explore interactions of its TonB box and determine whether additional FepA-TonB interaction sites exist. We found numerous specific sites of FepA interaction with TonB on the periplasmic face of the FepA cork in addition to the TonB box. Two residues, T32 and A33, might constitute a ligand-sensitive conformational switch. The facts that some interactions were enhanced in the absence of ligand and that other interactions did not require the TonB box argued against the current model and suggested that the transport process is more complex than originally conceived, with subtleties that might provide a mechanism for discrimination among ligand-loaded transporters. These results constitute the first study on the dynamics of TonB-gated transporter interaction with TonB in vivoIMPORTANCE The TonB system of Gram-negative bacteria has a noncanonical active transport mechanism involving signal transduction and proteins integral to both membranes. To achieve transport, the cytoplasmic membrane protein TonB physically contacts outer membrane transporters such as FepA. Only one contact between TonB and outer membrane transporters has been identified to date: the TonB box at the transporter amino terminus. The TonB box has low information content, raising the question of how TonB can discriminate among multiple different TonB-dependent transporters present in the bacterium if it is the only means of contact. Here we identified several additional sites through which FepA contacts TonB in vivo, including two neighboring residues that may explain how FepA signals to TonB that ligand has bound.

Project description: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 disulfide-linked 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.

Dataset Information

Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

Publications

Identification of functionally important TonB-ExbD periplasmic domain interactions in vivo.

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