In Gram-negative bacteria, outer membrane transporters import nutrients by coupling to an inner membrane protein complex called the Ton complex. The Ton complex consists of TonB, ExbB, and ExbD, and uses the proton motive force at the inner membrane to transduce energy to the outer membrane via TonB. Here, we structurally characterize the Ton complex from Escherichia coli using X-ray crystallography, electron microscopy, double electron-electron resonance (DEER) spectroscopy, and crosslinking. Our results reveal a stoichiometry consisting of a pentamer of ExbB, a dimer of ExbD, and at least one TonB. 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.

Structural insight into the role of the Ton complex in energy transduction

Structure and Function of Stator Units of the Bacterial Flagellar Motor

The TonB-ExbB-ExbD molecular motor harnesses the proton motive force across the bacterial inner membrane to couple energy to transporters at the outer membrane, facilitating uptake of essential nutrients such as iron and cobalamine. TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Until recently, no high-resolution structural information was available for this unique molecular motor. We published the first crystal structure of ExbB-ExbD in 2016 and showed that five copies of ExbB are arranged as a pentamer around a single copy of ExbD. However, our spectroscopic experiments clearly indicated that two copies of ExbD are present in the complex. To resolve this ambiguity, we used single-particle cryo-electron microscopy to show that the ExbB pentamer encloses a dimer of ExbD in its transmembrane pore, and not a monomer as previously reported. The revised stoichiometry has implications for motor function.

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Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 A-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction.

https://biorxiv.org/content/10.1101/2020.05.15.096610v1.full.pdf

Structure and function of stator units of the bacterial flagellar motor

Many biological processes that are essential for life are powered by the flow of ions across the membranes of cells. Similar to how energy is stored in the water behind a dam, energy is also stored when the concentration of ions on one side of a biological membrane is higher than it is on the other. When these ions then flow down this concentration gradient, the energy can be harnessed to power other processes. In many bacteria, the concentration of hydrogen ions, or protons, is higher on the outside of the cell. When the protons flow down the concentration gradient, a protein complex called the Ton system in the bacteria’s inner membrane harnesses the energy to transport various compounds, including essential nutrients, across the outer membrane, which is about 20 nanometres away. Toxins, and viruses that infect bacteria, can also hijack the Ton system to gain entry into these cells. This means that the Ton system could perhaps be targeted via drugs to treat bacterial infections. Though the Ton system is important, structural information on this protein family is limited. The Ton complex is composed of three proteins – ExbB, ExbD and TonB – located in the bacteria’s inner membrane. ExbB and ExbD together form a channel for the protons and the complex made from these two proteins can be thought of as the system’s engine. Maki-Yonekura et al. wanted to understand how the ExbB / ExbD complex works, which was challenging because the complex was not well suited to any single structural biology technique. To get around this issue, a combination of two techniques called X-ray crystallography and single particle cryo-EM were used. This approached revealed that the two proteins form complexes made up of either five or six ExbB subunits with one or three ExbD subunits, respectively. It also showed that the proteins transition between the two forms in a cell’s membrane. More of the larger six-unit complex (also called a “hexamer”) formed at higher pH. This is consistent with the increased flow of protons through the channel when the local conditions inside the cell become less acidic. Based on these results, Maki-Yonekura et al. propose that some subunits in the core of the complex rotate to harness the energy from the flow of protons, and the number of subunits in the complex changes when it switches to become active or inactive. The discoveries may provide a new vision of dynamic membrane biology. Further studies are now needed to see how general this mechanism is in biology, and the new structural information could also be used to help develop more anti-bacterial drugs.

Hexameric and pentameric complexes of the ExbBD energizer in the Ton system

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.

Kristian T. Levey

Papers

Membrane Protein Complex ExbB4-ExbD1-TonB1 from Escherichia coli Demonstrates Conformational Plasticity