title: Mutations in the ExbB cytoplasmic carboxy terminus prevent energy-dependent interaction between the TonB and ExbD periplasmic domains.
abstract: The TonB system of Gram-negative bacteria provides passage across the outer membrane (OM) diffusion barrier that otherwise limits access to large, scarce, or important nutrients. In Escherichia coli, the integral cytoplasmic membrane (CM) proteins TonB, ExbB, and ExbD couple the CM proton motive force (PMF) to active transport of iron-siderophore complexes and vitamin B(12) across the OM through high-affinity transporters. ExbB is an integral CM protein with three transmembrane domains. The majority of ExbB occupies the cytoplasm. Here, the importance of the cytoplasmic ExbB carboxy terminus (residues 195 to 244) was evaluated by cysteine scanning mutagenesis. D211C and some of the substitutions nearest the carboxy terminus spontaneously formed disulfide cross-links, even though the cytoplasm is a reducing environment. ExbB N196C and D211C substitutions were converted to Ala substitutions to stabilize them. Only N196A, D211A, A228C, and G244C substitutions significantly decreased ExbB activity. With the exception of ExbB(G244C), all of the substituted forms were dominant. Like wild-type ExbB, they all formed a formaldehyde cross-linked tetramer, as well as a tetramer cross-linked to an unidentified protein(s). In addition, they could be formaldehyde cross-linked to ExbD and TonB. Taken together, the data suggested that they assembled normally. Three of four ExbB mutants were defective in supporting both the PMF-dependent formaldehyde cross-link between the periplasmic domains of TonB and ExbD and the proteinase K-resistant conformation of TonB. Thus, mutations in a cytoplasmic region of ExbB prevented a periplasmic event and constituted evidence for signal transduction from cytoplasm to periplasm in the TonB system.
authors.affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
authors.forename: Bimal
authors.initials: B
authors.lastname: Jana
authors.forename: Marta
authors.initials: M
authors.lastname: Manning
authors.forename: Kathleen
authors.initials: K
authors.lastname: Postle
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D005557:Formaldehyde / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary
chemical_list: 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