Interactions in the TonB-Dependent Energy Transduction Complex: ExbB and ExbD Form Homomultimers

Abstract

The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane ofEscherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.

Quantification of known components of the Escherichia coli TonB energy transduction system: TonB, ExbB, ExbD and FepA

“…Although an ExbB-ExbD cross-linked complex has not been identified, ExbD co-purifies with His 6 -tagged ExbB (Braun et al, 1996), and the chemical stability of ExbD requires ExbB (Fischer et al, 1989), suggesting that these proteins interact. TonB can cross-link in vivo to both ExbB and ExbD (Larsen et al, 1994;Higgs et al, 1998). Based on these observations, and the expectation that ExbB and ExbD would be expressed at equimolar levels, we have previously proposed an arrangement in which a single TonB resided in complex with homotrimers of ExbB and homotrimers of ExbD (Higgs et al, 1998).…”

“…Little is known regarding the quaternary structure of the TonB energy transduction complex or the ratios of that complex to the TonB-gated transporters. Using in vivo cross-linking, ExbD was shown to cross-link efficiently into both homodimers and homotrimers (Higgs et al, 1998). ExbB was shown to cross-link into homodimers and a yet higher mass complex that appears to include three or four ExbB molecules and one or more unidentified protein(s) (Higgs et al, 1998).…”

“…Using in vivo cross-linking, ExbD was shown to cross-link efficiently into both homodimers and homotrimers (Higgs et al, 1998). ExbB was shown to cross-link into homodimers and a yet higher mass complex that appears to include three or four ExbB molecules and one or more unidentified protein(s) (Higgs et al, 1998). Although an ExbB-ExbD cross-linked complex has not been identified, ExbD co-purifies with His 6 -tagged ExbB (Braun et al, 1996), and the chemical stability of ExbD requires ExbB (Fischer et al, 1989), suggesting that these proteins interact.…”

The plug domain of a neisserial TonB‐dependent transporter retains structural integrity in the absence of its transmembrane β‐barrel

“…TonB contains a single membrane-spanning K-helix, which anchors it in the inner membrane, and a large periplasmic domain, which interacts with the outer membrane transporter. The integral inner membrane proteins ExbB and ExbD interact with TonB, and are required for optimal energy transduction [42]. Consistent with other TonB-dependent transporters, gonococcal TbpA has also been demonstrated to interact with TonB [22].…”

Extraction, Purification, and Identification of Yersiniabactin, the Siderophore of Yersinia pestis

“…In E. coli, enterbactin interacts with the OM receptor FepA. Enterobactin is then passed into the periplasm to FepB, which delivers it to the cytoplasmic pores formed by FepD and FeG (Larsen et al, 1994;Ferguson et al, 1998Ferguson et al, , 2002Higgs et al, 1998;Buchanan et al, 1999). These ferric-siderophore receptors are expressed during iron starvation conditions and generally found at low levels during iron sufficient periods (Neidhardt and Curtiss, 1996).…”