Cytoplasmic membrane protonmotive force energizes periplasmic interactions between ExbD and TonB.
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.
Ollis
Anne A
AA
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, PA 16802, USA.
Manning
Marta
M
Held
Kiara G
KG
Postle
Kathleen
K
D019943:Amino Acid Substitution; D003432:Cross-Linking Reagents; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D005557:Formaldehyde; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000378:metabolism; D018892:Proton-Motive Force
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D008565:Membrane Proteins; C489181:tonB protein, E coli; C060790:exbD protein, E coli; D005557:Formaldehyde