Cytoplasmic membrane protonmotive force energizes periplasmic interactions between ExbD and TonB.

Abstract:

:The TonB system of Escherichia coli (TonB/ExbB/ExbD) transduces the protonmotive force (pmf) of the cytoplasmic membrane to drive active transport by high-affinity outer membrane transporters. In this study, chromosomally encoded ExbD formed formaldehyde-linked complexes with TonB, ExbB and itself (homodimers) in vivo. Pmf was required for detectable cross-linking between TonB-ExbD periplasmic domains. Consistent with that observation, the presence of inactivating transmembrane domain mutations ExbD(D25N) or TonB(H20A) also prevented efficient formaldehyde cross-linking between ExbD and TonB. A specific site of periplasmic interaction occurred between ExbD(A92C) and TonB(A150C) and required functional transmembrane domains in both proteins. Conversely, neither TonB, ExbB nor pmf were required for ExbD dimer formation. These data suggest two possible models where either dynamic complex formation occurred through transmembrane domains or the transmembrane domains of ExbD and TonB configure their respective periplasmic domains. Analysis of T7-tagged ExbD with anti-ExbD antibodies revealed that a T7 tag was responsible both for our previous failure to detect T7-ExbD-ExbB and T7-ExbD-TonB formaldehyde-linked complexes and for the concomitant artefactual appearance of T7-ExbD trimers.

journal_name

Mol Microbiol

journal_title

Molecular microbiology

authors

Ollis AA,Manning M,Held KG,Postle K

doi

10.1111/j.1365-2958.2009.06785.x

subject

Has Abstract

pub_date

2009-08-01 00:00:00

pages

466-81

issue

3

eissn

0950-382X

issn

1365-2958

pii

MMI6785

journal_volume

73

pub_type

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