1.A.30 The H⁺- or Na⁺-translocating Bacterial Flagellar Motor/ExbBD Outer Membrane Transport Energizer (Mot-Exb) Superfamily

The Mot-Exb Superfamily consists of six distant families, each probably with a distinct physiological function, although all may function as H⁺/Na⁺ channels, driving an energy-requiring process. For example, flagellar motors of marine bacteria Halomonas are driven by both protons and sodium ions (Kita-Tsukamoto et al. 2004). The MotAB family energizes bacterial flagellar rotation while the ExbBD family energizes accumulation of large molecules (i.e. iron-siderophores, vitamin B₁₂, DNA from phage, and colicins) from the external medium across the outer Gram-negative bacterial membrane into the periplasm. The AglRS system powers gliding motility while the SilAB systems energize gian adhesin export.  The function of a 5th family (TC# 1.A.30.5) is not known, but the ZorAB systems have been reported to function as parts of antiphage defense systems. The pmf (or smf) is probably the driving force in all cases. MotAB and PomAB are homologous to ExbBD and TolQR. MotAB of E. coli, the stator, is known to form a proton channel. This stator is composed of MotA and MotB proteins, which form a hetero-hexameric complex with a stoichiometry of four MotA and two MotB molecules. MotA can form a tetramer in the absence of MotB (Takekawa et al. 2016). Ion binding residues for Na⁺ flow in the stator complex of the Vibrio flagellar motor have been identified (Onoue et al. 2019). The structure and dynamics of the bacterial flagellum have been reviewed (Nakamura and Minamino 2019). The flagellar motor, which structurally resembles an artificial motor, is embedded within the cell envelop and spins at several hundred revolutions per second (Morimoto and Minamino 2021). A single nucleotide polymorphism alters the activity of the renal Na⁺:Cl^- cotransporter and reveals a role for transmembrane segment 4 in chloride and thiazide affinity (Moreno et al. 2004).

About 10 stator (MotA/MotB complexes) are docked around a rotor, and stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators in Salmonella.  Suzuki et al. 2019 showed that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. Thus, MotA-Met206 is involved in torque generation and proton translocation, and stator assembly is stabilized by protonation of the stator. Ancestral sequence reconstructions of MotB require MotA and give rise to pmf-dependent motility (Islam et al. 2020). The flagellar motor consists of a rotor and multiple stator units, each of which couples the ion flow through its ion channel with force generation (Terahara et al. 2020). The flagellar building blocks and motor proteins are well conserved among bacterial species, but structural and functional diversity of flagella has been revealed. The structure and function of the flagellar motor of the Gram-positive bacterium, Bacillus subtilis, differ from those of E. coli and Salmonella. The flagellar motor of the B. subtilis BR151MA strain possesses two distinct types of stator complexes, H⁺-type MotAB and Na⁺-type MotPS, around the rotor. These two types of stator units dynamically assemble to and disassemble from the rotor in response to environmental changes such as viscosity and external Na⁺ concentrations (Terahara et al. 2020).

The stator complex, MotA₅-MotB₂ is the heptameric complex that forms an ion channel. It consists of a ring of five MotA subunits that rotate around a central dimer of MotB subunits (Ridone et al. 2023). TMS3 and TMS4 of MotA combine with the single TMS of MotB to form two separate ion channels within this complex. The ion binding site and ion specificity are known. Ridone et al. 2023 modelled the central MotB dimer using coiled-coil engineering and modelling principles and calculated free energies to identify stable states in the operating cycle of the stator. They found 3 stable coiled-coil states with dimer interface angles of 28 degrees , 56 degrees and 64 degrees. Strategic mutagenesis on the comparative energy of the states were examined, and the motility was correlated with a specific hierarchy of stability between the three states. The results indicated agreement with existing models describing a 36 degrees rotation step of the MotA pentameric ring during the power stroke and provide an energetic basis for the coordinated rotation of the central MotB dimer based on coiled-coil modelling (Ridone et al. 2023).  Zinke et al. 2024 have proposed a mechanistic model for the Ton system, emphasizing ExbD duality and the pivotal catalytic role of peptidoglycan. Sequence analysis suggests that this mechanism is conserved in other systems energizing gliding motility and membrane integrity.

Yonekura et al. (2011) presented the first three-dimensional structure of the PomAB torque-generating stator unit analyzed by electron microscopy. The structure of PomAB revealed two arm domains, which contain the PG-binding site, connected to a large base made of the transmembrane and cytoplasmic domains. The arms lean downward to the membrane surface, likely representing a 'plugged' conformation, which would prevent ions leaking through the channel. They proposed a model for how PomAB units are placed around the flagellar basal body to function as torque generators. CryoEM image analysis revealed the overall structure of the Salmonella flagellum, and this structural information made it possible to discuss flagellar assembly and function at the atomic level. Minamino and Kinoshita 2023 described what is known about the structure, assembly, and function of Salmonella flagella.

Gram-negative bacteria operate a multi-protein Ton system to transport essential nutrients like metals and vitamins. This system harnesses the proton motive force at the inner membrane to energize the import through the outer membrane.  The periplasmic domain of ExbD is a dynamic dimer switching between two conformations representing the proton channel's open and closed states. This conformational switch is essential for the nutrient uptake by bacteria. The open state of ExbD triggers a disorder to order transition of TonB, enabling TonB to supply energy to the nutrient transporter. The peptidoglycan layer plays an anchoring function in this mechanism (Zinke et al. 2024).

Leu46 of MotB acts as the gate for hydronium ion permeation, which induces the formation of a water wire that may mediate the proton transfer to Asp32 on MotB. The free energy barrier for H₃O⁺ permeation is consistent with the proton transfer rate deduced from the flagellar rotational speed and the number of protons per rotation, suggesting that gating is the rate-limiting step (Nishihara and Kitao 2015). Structure and dynamics of MotA/B with nonprotonated and protonated Asp32 suggested size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintains a kink whereas protonation induces a straighter shape. Assuming that the cytoplasmic domain not included in the atomic model moves as a rigid body, the protonation/deprotonation of Asp32 is inferred to induce a ratchet motion of the cytoplasmic domain, correlated with the motion of the flagellar rotor (Nishihara and Kitao 2015). The rotational motor, located at the base of the flagellum, is the stator unit complex which conducts cations such as protons (H⁺) and sodium ions (Na⁺) down their electrochemical potentials across the cytoplasmic membrane and interacts with the rotor to generate the rotational force. Escherichia coli and Salmonella have the H⁺-type stator complex, which serves as a transmembrane H⁺ channel that couples H⁺ flow through this channel driving torque generation, whereas Vibrio and some Bacillus species have the Na⁺-type stator complex. Morimoto and Minamino 2023 described how to measure the ion conductivity of the transmembrane stator complex over-expressed in E. coli cells using fluorescent indicators. Intensity measurements of fluorescent indicators using either a fluorescence spectrophotometer or microscope allow quantitative detection of changes in the intracellular ion concentrations due to the ion channel activity of the transmembrane protein complex.

ExbBD forms both hexameric and pentameric complexes that coexist, with the proportion of the hexamers increasing with pH. Channel current measurements and 2D crystallography thus support the existence of and transition between the two oligomeric states in membranes. The hexameric complex has been reported to consist of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel (Maki-Yonekura et al. 2018). TonB physically interacts with the nutrient-loaded transporter to exert a force that opens an import pathway across the outer membrane. Another group showed that five copies of ExbB are arranged as a pentamer around two copies of ExbD in the complex. The revised stoichiometry has implications for motor function (Celia et al. 2019).

As noted above, each flagellum is a supramolecular motility machine consisting of a bi-directional rotary motor, a universal joint and a helical propeller. The signal transducers transmit environmental signals to the flagellar motor through the cytoplasmic chemotactic signaling pathway. The flagellar motor is composed of a rotor and multiple stator units, each of which acts as a transmembrane proton channel to conduct protons and exert force on the rotor (Minamino et al. 2019). FliG, FliM and FliN form the C ring on the cytoplasmic face of the basal body MS ring made of the transmembrane protein FliF and act as the rotor. The C ring also serves as a switching device that enables the motor to spin in both counterclockwise (CCW) and clockwise (CW) directions. The phosphorylated form of the chemotactic signaling protein CheY binds to FliM and FliN to induce conformational changes of the C ring responsible for switching the direction of flagellar motor rotation from CCW to CW (Minamino et al. 2019).