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ExbD mutants define initial stages in TonB energization.

ABSTRACT: Cytoplasmic membrane proteins ExbB and ExbD of the Escherichia coli TonB system couple cytoplasmic membrane protonmotive force (pmf) to TonB. TonB transmits this energy to high-affinity outer membrane active transporters. ExbD is proposed to catalyze TonB conformational changes during energy transduction. Here, the effect of ExbD mutants and changes in pmf on TonB proteinase K sensitivity in spheroplasts was examined. Spheroplasts supported the pmf-dependent formaldehyde cross-link between periplasmic domains of TonB and ExbD, indicating that they constituted a biologically relevant in vivo system to study changes in TonB proteinase K sensitivity. Three stages in TonB energization were identified. In Stage I, ExbD L123Q or TonB H20A prevented proper interaction between TonB and ExbD, rendering TonB sensitive to proteinase K. In Stage II, ExbD D25N supported conversion of TonB to a proteinase-K-resistant form, but not energization of TonB or formation of the pmf-dependent formaldehyde cross-link. Addition of protonophores had the same effect as ExbD D25N. This suggested the existence of a pmf-independent association between TonB and ExbD. TonB proceeded to Stage III when pmf was present, again becoming proteinase K sensitive, but now able to form the pmf-dependent cross-link to ExbD. Absence or presence of pmf toggled TonB between Stage II and Stage III conformations, which were also detected in wild-type cells. ExbD also underwent pmf-dependent conformational changes that were interdependent with TonB. These observations supported the hypothesis that ExbD couples TonB to the pmf, with concomitant transitions of ExbD and TonB periplasmic domains from unenergized to energized heterodimers.

SUBMITTER: Ollis AA

PROVIDER: S-EPMC3258360 | biostudies-other | 2012 Jan

REPOSITORIES: biostudies-other

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ExbD mutants define initial stages in TonB energization.

Ollis Anne A AA Postle Kathleen K

Journal of molecular biology 20111109 2

Cytoplasmic membrane proteins ExbB and ExbD of the Escherichia coli TonB system couple cytoplasmic membrane protonmotive force (pmf) to TonB. TonB transmits this energy to high-affinity outer membrane active transporters. ExbD is proposed to catalyze TonB conformational changes during energy transduction. Here, the effect of ExbD mutants and changes in pmf on TonB proteinase K sensitivity in spheroplasts was examined. Spheroplasts supported the pmf-dependent formaldehyde cross-link between perip ...[more]

PMID: 22100395

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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.

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ExbD mutants define initial stages in TonB energization.

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ExbD mutants define initial stages in TonB energization.

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