In Vivo Evidence for TonB Dimerization

Abstract

TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and 80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

Characterization of the Outer Membrane Receptor ShuA from the Heme Uptake System of Shigella dysenteriae

“…Furthermore, it is apparent that ShuA can interact with TonB in either the holo-or apo-form, as has previously been noted for the ferric hydroxamate receptor FhuA and truncated forms of TonB (52). Additionally, the interaction of ShuA with the full-length TonB yields a 1:1 complex, in contrast to previous reports in which an in vivo bacterial two-hybrid system suggested TonB exists as dimer (53). It was further reported that the dimerization required the N-terminal cytoplasmic membrane anchor as well as the periplasmic C-terminal domain.…”

Colicin Biology

“…First, the ability of ToxR-TonB hybrid proteins to activate the transcription of lacZ under the control of the cholera toxin promoter demonstrated ExbB/ExbD-dependent dimerization of TonB through the transmembrane domain. In contrast, the carboxy-terminal domain (residues 164 through 239) was capable of mediating dimerization as well but without the need for ExbB/ExbD, perhaps mimicking what occurs in the crystal/ NMR structures (573). Second, with the exception of F180C, cysteine replacements at each of the carboxy-terminal aromatic residues form spontaneous disulfide-linked dimers that leave TonB confined to the IM.…”

Crystal Structure of a 92-Residue C-terminal Fragment of TonB from Escherichia coli Reveals Significant Conformational Changes Compared to Structures of Smaller TonB Fragments

“…Under in vivo conditions Sauter et al (46) using ToxR fusions, found that the periplasmic part of TonB existed as a monomer, whereas the entire TonB and the C-terminal TonB-76 fragment formed dimers. Under in vitro conditions Koedding et al (21) with analytical ultracentrifugation experiments found that TonB-77 and TonB-85 formed dimers, whereas longer TonB fragments up to TonB-116 formed monomers.…”