Mutations in the ExbB Cytoplasmic Carboxy Terminus Prevent Energy-Dependent Interaction between the TonB and ExbD Periplasmic Domains

Jana, Bimal; Manning, Marta; Postle, Kathleen

doi:10.1128/jb.05674-11

Cited by 21 publications

(60 citation statements)

References 50 publications

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“…By single-particle electron microscopy (EM), we noted occasional periplasmic homodimerization by ExbD that correlated with two different arrangements of ExbB's cytoplasmic domains: a tetrameric arrangement and a dimer of dimers. Despite the lack of a functional assay, our results are consistent with in vivo data (18,22,26). Our biophysical analyses were expanded by the use of amphipols (47).…”

supporting

confidence: 76%

“…Considering in vivo protein quantitation (44), we propose that a second molecule of ExbD occupies the place of TonB. This coordination could be mediated by multifunctional or essential housekeeping proteins that have been noted by in vivo cross-linking to ExbB (18,22) and to ExbD (15, , and no observed dimerization on the periplasmic side of the micelle (C). Volumes in panels B and C show four regions of density descending from the micelle, which can be attributed to a tetramer of ExbB.…”

Section: Discussionmentioning

confidence: 99%

“…Apparently, no ExbB residue directly participates in proton translocation across the CM (18). Rather, ExbB has a structural "scaffolding" role, assembling into dimers and tetramers (22) and potentially propagating signals between the cytoplasm and the periplasm (18).…”

mentioning

confidence: 99%

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Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity

et al. 2015

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Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His 6 -tagged exbB-exbD and S-tagged tonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ϳ230 kDa with a hydrodynamic radius of ϳ6.0 nm were similar to previously purified ExbB 4 -ExbD 2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB 4 -ExbD 1 -TonB 1 . Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB 4 -ExbD 1 -TonB 1 . Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbBExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization. IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB 4 -ExbD 1 -TonB 1 complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

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confidence: 76%

Section: Discussionmentioning

confidence: 99%

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Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity

et al. 2015

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“…ExbD⌬12-21 also failed to support the conformational response of TonB to PMF. Recently, residues in the cytoplasmic carboxy terminus of ExbB, where contact with the cytoplasmic amino terminus of ExbD is possible, were also shown to be important for the response of the ExbD and TonB periplasmic domains to PMF (16). Residues 12 to 17 do not appear to be essential for ExbD function because ExbD⌬4-15, ExbD-H16A, and ExbD-D17A retain activity (reference 2 and data not shown).…”

Section: Discussionmentioning

confidence: 99%

The ExbD Periplasmic Domain Contains Distinct Functional Regions for Two Stages in TonB Energization

2012

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The TonB system of Gram-negative bacteria energizes the active transport of diverse nutrients through high-affinity TonB-gated outer membrane transporters using energy derived from the cytoplasmic membrane proton motive force. Cytoplasmic membrane proteins ExbB and ExbD harness the proton gradient to energize TonB, which directly contacts and transmits this energy to ligand-loaded transporters. In Escherichia coli , the periplasmic domain of ExbD appears to transition from proton motive force-independent to proton motive force-dependent interactions with TonB, catalyzing the conformational changes of TonB. A 10-residue deletion scanning analysis showed that while all regions except the extreme amino terminus of ExbD were indispensable for function, distinct roles for the amino- and carboxy-terminal regions of the ExbD periplasmic domain were evident. Like residue D25 in the ExbD transmembrane domain, periplasmic residues 42 to 61 facilitated the conformational response of ExbD to proton motive force. This region appears to be important for transmitting signals between the ExbD transmembrane domain and carboxy terminus. The carboxy terminus, encompassing periplasmic residues 62 to 141, was required for initial assembly with the periplasmic domain of TonB, a stage of interaction required for ExbD to transmit its conformational response to proton motive force to TonB. Residues 92 to 121 were important for all three interactions previously observed for formaldehyde-cross-linked ExbD: ExbD homodimers, TonB-ExbD heterodimers, and ExbD-ExbB heterodimers. The distinct requirement of this ExbD region for interaction with ExbB raised the possibility of direct interaction with the few residues of ExbB known to occupy the periplasm.

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“…Previously, we engineered Cys substitutions in each of two TonB system proteins to identify sites of interaction via disulfide cross-linking (49)(50)(51)(52)(53)(54). Because the exact sites of interaction between the two proteins are unknown, many combinations of cysteine substitutions must be engineered in order to map the interaction interface.…”

Section: Discussionmentioning

confidence: 99%

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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Mutations in the ExbB Cytoplasmic Carboxy Terminus Prevent Energy-Dependent Interaction between the TonB and ExbD Periplasmic Domains

Cited by 21 publications

References 50 publications

Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity

Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity

The ExbD Periplasmic Domain Contains Distinct Functional Regions for Two Stages in TonB Energization

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

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Mutations in the ExbB Cytoplasmic Carboxy Terminus Prevent Energy-Dependent Interaction between the TonB and ExbD Periplasmic Domains

Cited by 21 publications

References 50 publications

Membrane Protein Complex ExbB 4 -ExbD 1 -TonB 1 from Escherichia coli Demonstrates Conformational Plasticity

Membrane Protein Complex ExbB 4 -ExbD 1 -TonB 1 from Escherichia coli Demonstrates Conformational Plasticity

The ExbD Periplasmic Domain Contains Distinct Functional Regions for Two Stages in TonB Energization

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

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Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity

Membrane Protein Complex ExbB ₄ -ExbD ₁ -TonB ₁ from Escherichia coli Demonstrates Conformational Plasticity