Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity
Aleksandr Sverzhinsky,Jacqueline W. Chung,Justin C. Deme,Lucien Fabre,Kristian T. Levey,Maria Plesa,David M. Carter,Patrick Lypaczewski,James W. Coulton +8 more
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In vitro evidence is demonstrated of the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex, and structural biology that provides insights into 3D organization are presented.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 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.read more
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Structural insight into the role of the Ton complex in energy transduction
Herve Celia,Nicholas Noinaj,Stanislav D. Zakharov,Enrica Bordignon,Istvan Botos,Monica Santamaria,Travis J. Barnard,William A. Cramer,Roland Lloubès,Susan K. Buchanan +9 more
TL;DR: Electrophysiology studies show that the Ton subcomplex forms pH-sensitive cation-selective channels and provide insight into the mechanism by which it may harness the proton motive force to produce energy.
Journal ArticleDOI
Structure and Function of Stator Units of the Bacterial Flagellar Motor
Mònica Santiveri,Aritz Roa-Eguiara,Caroline Kühne,Navish Wadhwa,Haidai Hu,Howard C. Berg,Marc Erhardt,Nicholas M.I. Taylor +7 more
TL;DR: This work identifies key residues involved in torque generation and presents a detailed mechanistic model for motor function and switching of rotational direction in the stator unit.
Journal ArticleDOI
Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry
Herve Celia,Istvan Botos,Xiaodan Ni,Tara Fox,Tara Fox,Natalia de Val,Natalia de Val,Roland Lloubès,Jiansen Jiang,Susan K. Buchanan +9 more
TL;DR: The single-particle cryo-EM structure of the bacterial ExbB–ExbD subcomplex reconstituted in lipid nanodiscs is reported, showing that the Ex bB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported, with implications for motor function.
Posted ContentDOI
Structure and function of stator units of the bacterial flagellar motor
Mònica Santiveri,Aritz Roa-Eguiara,Caroline Kühne,Navish Wadhwa,Howard C. Berg,Marc Erhardt,Nicholas M.I. Taylor +6 more
TL;DR: This work identifies key residues involved in torque generation and presents a mechanistic model for motor function and switching of rotational direction in the bidirectional bacterial flagellar motor.
Journal ArticleDOI
Hexameric and pentameric complexes of the ExbBD energizer in the Ton system
Saori Maki-Yonekura,Rei Matsuoka,Yoshiki Yamashita,Hirofumi Shimizu,Maiko Tanaka,Fumie Iwabuki,Koji Yonekura +6 more
TL;DR: This work presents structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM and proposes models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.
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