Membrane Protein Complex ExbB 4 -ExbD 1 -TonB 1 from Escherichia coli Demonstrates Conformational Plasticity
| Autor: | Maria Plesa, Aleksandr Sverzhinsky, Justin C. Deme, Jacqueline W. Chung, Kristian T. Levey, Patrick Lypaczewski, Lucien Fabre, James W. Coulton, David M. Carter |
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| Rok vydání: | 2015 |
| Předmět: |
Phage display
Chemiosmosis Escherichia coli Proteins Cell Membrane Membrane Proteins Articles Plasma protein binding Periplasmic space Biology Crystallography X-Ray Microbiology Cell membrane medicine.anatomical_structure Biochemistry Structural biology Membrane protein complex Periplasm Escherichia coli medicine bacteria Bacterial outer membrane Dimerization Molecular Biology Protein Binding |
| Zdroj: | Journal of Bacteriology. 197:1873-1885 |
| ISSN: | 1098-5530 0021-9193 |
| DOI: | 10.1128/jb.00069-15 |
| Popis: | 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 His 6 -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 ExbB 4 -ExbD 2 complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB 4 -ExbD 1 -TonB 1 . 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 ExbB 4 -ExbD 1 -TonB 1 . 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. IMPORTANCE 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 ExbB 4 -ExbD 1 -TonB 1 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. |
| Databáze: | OpenAIRE |
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