title: Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity.
abstract: Iron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-tagged exbB-exbD and S-tagged tonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB from Escherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB₄-ExbD₂ complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB₄-ExbD₁-TonB₁. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB₄-ExbD₁-TonB₁. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.
  
  
  OBJECTIVE
  Receptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstrate in vitro evidence of ExbB₄-ExbD₁-TonB₁ complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Aleksandr
authors: A
authors: Sverzhinsky
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Jacqueline W
authors: JW
authors: Chung
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Justin C
authors: JC
authors: Deme
authors: Department of Anatomy and Cell Biology, McGill University, Montreal, QC, Canada.
authors: Lucien
authors: L
authors: Fabre
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Kristian T
authors: KT
authors: Levey
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Maria
authors: M
authors: Plesa
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: David M
authors: DM
authors: Carter
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada.
authors: Patrick
authors: P
authors: Lypaczewski
authors: Department of Microbiology and Immunology, McGill University, Montreal, QC, Canada Microbiome and Disease Tolerance Centre, McGill University, Montreal, QC, Canada james.coulton@mcgill.ca.
authors: James W
authors: JW
authors: Coulton
mesh_terms: D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019897:Periplasm / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D029968:Escherichia coli Proteins; C052874:ExbB protein, E coli; D008565:Membrane Proteins; C489181:tonB protein, E coli; C060790:exbD protein, E coli