ExbD mutants define initial stages in TonB energization

Anne A. Ollis, Kathleen Postle

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)237-247
Number of pages11
JournalJournal of Molecular Biology
Volume415
Issue number2
DOIs
StatePublished - Jan 13 2012

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Endopeptidase K
Spheroplasts
Formaldehyde
Cell Membrane
Membrane Transport Proteins
Membrane Proteins
Escherichia coli

All Science Journal Classification (ASJC) codes

  • Molecular Biology

Cite this

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title = "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.",
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ExbD mutants define initial stages in TonB energization. / Ollis, Anne A.; Postle, Kathleen.

In: Journal of Molecular Biology, Vol. 415, No. 2, 13.01.2012, p. 237-247.

Research output: Contribution to journalArticle

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AU - Postle, Kathleen

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AB - 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.

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