title: Coordinated rearrangements between cytoplasmic and periplasmic domains of the membrane protein complex ExbB-ExbD of Escherichia coli.
abstract: Gram-negative bacteria rely on the ExbB-ExbD-TonB system for the import of essential nutrients. Despite decades of research, the stoichiometry, subunit organization, and mechanism of action of the membrane proteins of the Ton system remain unclear. We copurified ExbB with ExbD as an ∼240 kDa protein-detergent complex, measured by light scattering and by native gels. Quantitative Coomassie staining revealed a stoichiometry of ExbB4-ExbD2. Negative stain electron microscopy and 2D analysis showed particles of ∼10 nm diameter in multiple structural states. Nanogold labeling identified the position of the ExbD periplasmic domain. Random conical tilt was used to reconstruct the particles in three structural states followed by sorting of the single particles and refinement of each state. The different states are interpreted by coordinated structural rearrangements between the cytoplasmic domain and the periplasmic domain, concordant with in vivo predictions.
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Aleksandr
authors.initials: A
authors.lastname: Sverzhinsky
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Lucien
authors.initials: L
authors.lastname: Fabre
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Cottreau
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Damien M P
authors.initials: DM
authors.lastname: Biot-Pelletier
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Sofia
authors.initials: S
authors.lastname: Khalil
authors.affiliation: Facility for Electron Microscope Research, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Mihnea
authors.initials: M
authors.lastname: Bostina
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, QC H3A 2B4, Canada.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Rouiller
authors.affiliation: Department of Microbiology and Immunology, McGill University, Montreal, QC H3A 2B4, Canada; Microbiome and Disease Tolerance Centre, McGill University, Montreal, QC H3A 2B4, Canada. Electronic address: james.coulton@mcgill.ca.
authors.forename: James W
authors.initials: JW
authors.lastname: Coulton
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D007091:Image Processing, Computer-Assisted / Q000379:methods; D008854:Microscopy, Electron; D017434:Protein Structure, Tertiary
chemical_list: D003902:Detergents; D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; C060790:exbD protein, E coli