Studies on colicin B translocation: FepA is gated by TonB

Devanathan, Surendranathan; Postle, Kathleen

doi:10.1111/j.1365-2958.2007.05808.x

Cited by 49 publications

(76 citation statements)

References 57 publications

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“…It is unknown if these specific TonB-BtuB interactions occur in vivo, but this might identify some common TonB interactive sites that are shared with both ExbDand TonB-gated transporters. It is known that the TonB box is not the only site through which TonB interacts with the transporters in vivo, although specific sites remain to be determined (10). Two TonB regions, from 158 to 163 and 226 to 233, exhibit extensive in vitro interactions with residues of the BtuB TonB box (residues 6 to 12) in the cocrystal structure (51).…”

Section: Discussionmentioning

confidence: 99%

Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

Ollis

Postle

2012

J Bacteriol

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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.

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Section: Discussionmentioning

confidence: 99%

Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

Ollis

Postle

2012

J Bacteriol

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“…Our hypothesis is that the TonB carboxy-terminal domain can adopt several conformations in order to bind to structurally diverse outer membrane targets, but only the correct conformations elicited by prior interaction with the ExbD periplasmic domain will bring about correct contacts with transporters that lead to energy transduction (6). Our data suggest that other, yet-to-be-discovered important transporter-TonB interactions exist (29). This idea was also proposed years ago by Kadner and colleagues based on the low degree of structural discrimination to be found in TonB boxes (16).…”

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confidence: 87%

“…Furthermore, both in vivo and in vitro studies have detected significant movement of only roughly the amino-terminal one-third of the cork residues that are accessible from the periplasm. While removing one-third of the cork residues from the barrel would leave an opening sufficient for passage of transport ligands such as siderophores and vitamin B 12 , it may not be sufficient for larger ligands such as the 55-kDa protein toxin colicin B (24,(29)(30)(31)(32).…”

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Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

Gresock

Postle

2017

J Bacteriol

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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 ϳ158 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 ligandsensitive 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 vivo. IMPORTANCE 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 TonBdependent 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.KEYWORDS TonB, FepA, cork domain, p-benzoyl-L-phenylalanine, enterochelin, TonB box, energy transduction I ron is essential for growth of Gram-negative bacteria but can be difficult to acquire because of its insolubility under aerobic conditions, its scarcity in hosts, and the protective nature of the Gram-negative bacterial outer membrane (OM) (1). The majority of Gram-negative bacteria synthesize and secrete siderophores, which are high-

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“…We believe that 4 M urea (but not 3 M urea or less) partially or fully unfolds the N-terminal part of the protein that forms the plug of these channels, thereby opening an ion-conducting pathway. It is generally assumed that during transport (of siderophores or colicins) the plug domain either comes out of its barrel (11,25) or rearranges within the Fig. 6.…”

Section: Discussionmentioning

confidence: 99%

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

Udho

Jakes

Buchanan

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Micronutrients such as siderophore-bound iron and vitamin B12 cross the outer membrane of Gram-negative bacteria through a group of 22-stranded ␤-barrel proteins. They share the unusual feature that their N-terminal end inserts from the periplasmic side into the ␤-barrel and plugs the lumen. Transport results from energy-driven movement of TonB protein, which either pulls the plug out of the barrel or causes it to rearrange within the barrel. Attempts to reconstitute native plugged channels in an ionconducting state in lipid bilayer membranes have so far been unsuccessful. We, however, have discovered that if the cis solution contained 4 M urea, then, with the periplasmic side of the channel facing that solution, macroscopic conductances and single channel events could be observed. These results were obtained with FhuA, Cir, and BtuB; for the former two, the channels were closed by removing the 4 M urea. Channels generated by 4 M urea exposure were not a consequence of general protein denaturation, as their ligand-binding properties were preserved. Thus, with FhuA, addition of ferrichrome (its siderophore) to the trans, extracellularfacing side reversibly inhibited 4 M urea-induced channel opening and blocked the channels. With Cir, addition of colicin Ia (the microbial toxin that targets Cir) to the trans, extracellular-facing side prevented 4 M urea-induced channel opening. We hypothesize that 4 M urea reversibly unfolds the FhuA and Cir plugs, thereby opening an ion-conducting pathway through these channels, and that this mimics to some extent the in vivo action of TonB on these plugs.BtuB ͉ Cir ͉ FhuA ͉ urea-denaturation

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Studies on colicin B translocation: FepA is gated by TonB

Cited by 49 publications

References 57 publications

Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

Identification of Functionally Important TonB-ExbD Periplasmic Domain Interactions In Vivo

Going Outside the TonB Box: Identification of Novel FepA-TonB Interactions In Vivo

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

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