Oligomeric Structure of ExbB and ExbB-ExbD Isolated from <i>Escherichia coli</i> As Revealed by LILBID Mass Spectrometry

Pramanik, Avijit; Hauf, Waldemar; Hoffmann, Jan; Cernescu, Mihaela; Brutschy, Bernhard; Braun, Volkmar

doi:10.1021/bi2008195

Cited by 36 publications

(37 citation statements)

References 34 publications

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“…TonB was also not required for the formation of the ExbD 2 -ExbB complex. It should be noted that Pramanik et al recently identified an ExbB 6 -ExbD 1 complex in vitro and could find no evidence for ExbD dimers (42). In that study, however, ExbD carried a StrepII affinity tag at its carboxy terminus and ExbB carried a His 6 tag.…”

Section: Discussionmentioning

confidence: 69%

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Ollis

Postle

2011

J Bacteriol

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The TonB system couples cytoplasmic membrane proton motive force to TonB-gated outer membrane transporters for active transport of nutrients into the periplasm. In Escherichia coli, cytoplasmic membrane proteins ExbB and ExbD promote conformational changes in TonB, which transmits this energy to the transporters. The only known energy-dependent interaction occurs between the periplasmic domains of TonB and ExbD. This study identified sites of in vivo homodimeric interactions within ExbD periplasmic domain residues 92 to 121. ExbD was active as a homodimer (ExbD 2 ) but not through all Cys substitution sites, suggesting the existence of conformationally dynamic regions in the ExbD periplasmic domain. A subset of homodimeric interactions could not be modeled on the nuclear magnetic resonance (NMR) structure without significant distortion. Most importantly, the majority of ExbD Cys substitutions that mediated homodimer formation also mediated ExbD-TonB heterodimer formation with TonB A150C. Consistent with the implied competition, ExbD homodimer formation increased in the absence of TonB. Although ExbD D25 was not required for their formation, ExbD dimers interacted in vivo with ExbB. ExbD-TonB interactions required ExbD transmembrane domain residue D25. These results suggested a model where ExbD 2 assembled with ExbB undergoes a transmembrane domain-dependent transition and exchanges partners in localized homodimeric interfaces to form an ExbD 2 -TonB heterotrimer. The findings here were also consistent with our previous hypothesis that ExbD guides the conformation of the TonB periplasmic domain, which itself is conformationally dynamic.The TonB system of Gram-negative bacteria couples the proton motive force (PMF) of the cytoplasmic membrane (CM) to the active transport of a diverse range of large, scarce, or important nutrients across the unenergized outer membrane (1,24,25). Active transport across the outer membrane requires TonB-gated transporters, which are 22-stranded ␤-barrels with lumens occluded by an amino-terminal globular domain called the cork (37).In addition to the TonB-gated transporters in the outer membrane, the TonB system consists of 3 integral CM proteins, TonB, ExbB, and ExbD. TonB and ExbD have identical topologies, each with a single transmembrane domain (TMD) and more than two-thirds of each protein in the periplasm (15,20,43). ExbB has 3 TMDs and significant cytoplasmic domains (21,22). ExbB and ExbD appear to couple the energy of the CM PMF to conformational changes in the TonB carboxy terminus (33). The TonB carboxy terminus directly contacts the TonB-gated transporters and somehow transmits energy for active transport of substrates. Evidence suggests all three proteins form a complex in the CM, but the stoichiometry of this complex is unknown (2, 38). The cellular TonB/ExbD/ ExbB ratio is 1:2:7 (16).The only step in TonB energization currently known to require the PMF is characterized by a formaldehyde-cross-linkable interaction between the TonB and ExbD periplasmic domains. While Ton...

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Section: Discussionmentioning

confidence: 69%

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Ollis

Postle

2011

J Bacteriol

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“…1a). The exact stoichiometry of components of the Ton complex has been a matter of debate for years [16][17][18][19] . Evidence favoring a dynamic mechanism has been reported in which fluorescence anisotropy studies showed that the presence of TonB within the Ton complex sustains a rotational motion dependent on the pmf at the IM 20 .…”

Section: Introductionmentioning

confidence: 99%

Structural Insight into the Role of the Ton Complex in Energy Transduction

Celia

Noinaj

Zakarov

et al. 2018

Biophysical Journal

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SummaryIn Gram-negative bacteria, outer membrane (OM) transporters import nutrients by coupling to an inner membrane (IM) protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force (pmf) at the IM to transduce energy to the OM via TonB. Here, we structurally characterize the Ton complex from E. coli using X-ray crystallography, electron microscopy, DEER spectroscopy, and crosslinking, revealing a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

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“…The masses of E. coli TonB, ExbB, and ExbD are 26, 26, and 16 kDa, respectively. The stoichiometry of the TonB-ExbB-ExbD complex (1:6:1 [83], 1:4:1 [85], or 1:4:2 [41,84]) implies that the TonB component, with about one-fifth of the mass and considerably less inertia, will spin faster than the ExbBD component. It is relevant that both ExbB and ExbD contain sequence or structural homology to the PG-binding LysM domain (see Fig.…”

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confidence: 99%

“…Sverzhinsky et al (85) purified TonB-ExbBD with an apparent stoichiometry of 1:4:1. Considering that Pramanik et al (83) previously determined the TonB-ExbBD ratios to be 1:6:1, the exact composition of TonB-ExbBD remains uncertain, but it is likely that multiple copies of ExbB exist in the complex. Neither article (reference 41 or 85) considered TonB's affinity for PG nor the energy-dependent nanosecond-scale anisotropy of TonB-GFP (38), so the mechanisms they propose are difficult to reconcile with these aspects of TonB biochemistry.…”

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confidence: 99%

ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition

Klebba

2016

J Bacteriol

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.g., OmpA), localizing it near the periplasmic interface of the OM bilayer. Porins and other OM proteins associate with PG, and this general affinity allows the TonB CTD dimer to survey the periplasmic surface of the OM bilayer. Energized rotational motion of the TonB N terminus in the fluid IM bilayer promotes the lateral movement of the TonB-ExbBD complex in the IM and of the TonB CTD dimer across the inner surface of the OM. When it encounters an accessible TonB box of a (ligand-bound) LGP, the monomeric form of the CTD binds and recruits it into a 4-stranded ␤-sheet. Because the CTD is rotating, this binding reaction transfers kinetic energy, created by the electrochemical proton gradient across the IM, through the periplasm to the OM protein. The equilibration of the TonB C terminus between the dimeric and monomeric forms that engage in different binding reactions allows the identification of ironloaded LGP and then the internalization of iron through their trans-outer membrane ␤-barrels. Hence, the ROSET model postulates a mechanism for the transfer of energy from the IM to the OM, triggering iron uptake.T he 239-amino-acid TonB protein underlies several aspects of Gram-negative bacterial cell envelope physiology, including obligatory involvement in metal (iron) transport through the outer membrane (OM), susceptibility to numerous bacteriocins/ microcins, and infection by certain bacteriophages. Regarding the role in iron transport, bacteria generally acquire iron by the elaboration of siderophores (1) that complex it in the environment. The subsequent active transport of ferric siderophores through OM receptor proteins (2, 3) requires TonB (4, 5).FepA is one such OM protein that selectively recognizes and internalizes the native siderophore of Escherichia coli, ferric enterobactin (FeEnt) (6). The N-terminal 150-residue globular portion of FepA (N-domain) resides within a C-terminal 22-stranded ␤-barrel (Fig. 1). The C-terminal ␤-barrel places FepA and its relatives in the porin superfamily (7,8). Their selectivity for ligands (9-11) and the fact that high-affinity ligand binding (12) triggers uptake through their transmembrane channels led to the designation ligand-gated porins (LGP) (13). They require energy and TonB for functionality, so they are also energy-and TonBdependent receptors or TonB-dependent transporters (TBDT) (14). None of these designations is fully appropriate, in that OM proteins in this class are not diffusive porins (15) and neither are they true transporters, a term usually reserved for ATP-or proton motive force (PMF)-driven inner membrane (IM) permeases (16). With the additional stipulation that their activities are TonB and energy dependent, in this paper, I will refer to FepA and its orthologs/paralogs as LGP.The need for TonB in the energy-dependent uptake of metals (ferric siderophores [3], heme [17], and cobalt as vitamin B 12 [2]) is a key aspect of cellular nutrition and an enigmatic feature of cell envelope physiology. FeEnt transport through the OM requires the electroche...

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Oligomeric Structure of ExbB and ExbB-ExbD Isolated from Escherichia coli As Revealed by LILBID Mass Spectrometry

Cited by 36 publications

References 34 publications

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

The Same Periplasmic ExbD Residues Mediate In Vivo Interactions between ExbD Homodimers and ExbD-TonB Heterodimers

Structural Insight into the Role of the Ton Complex in Energy Transduction

ROSET Model of TonB Action in Gram-Negative Bacterial Iron Acquisition

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