Torque and switching in the bacterial flagellar motor. An electrostatic model

104

The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. where V m is the membrane voltage and Δμ = k B T ln(C in /C out ) and q are the transmembrane chemical potential difference and charge of the ions, respectively, with C in and C out the internal and external ion concentrations. In most species the primary form of biological free energy is the proton-motive force (PMF), the IMF for H + ions (1). Physiological PMF is typically in the range −150 mV to −200 mV, with the inside electrically negative and slightly alkaline relative to the outside. Some organisms use sodium-motive force (SMF) to drive numerous cellular processes, such as bacterial motility (2), ATP synthesis (3), and active membrane transport (4). Arguably the most important process driven by IMF is ATP synthesis, which generates cellular ATP by forced rotation of the F 1 part of F 1 F O ATP-synthase. F 1 is mechanically coupled to and rotated by F O , which like the bacterial flagellar motor (BFM) is an ion-driven rotary motor. Understanding the mechanism of these and other ion-driven molecular machines is a fundamental challenge in cellular energetics and biophysics. The BFM (Fig. 1A) is a rotary molecular machine that propels many species of swimming bacteria. It couples ion flow, for example protons (H + ) in Escherichia coli or sodium ions (Na + ) in Vibrio alginolyticus, to the rotation of extracellular helical flagellar filaments at hundreds of revolutions per second (Hz) (2, 5, 6). Torque is generated by interactions between stator complexes (containing the proteins MotA and MotB in E. coli and PomA and PomB in V. alginolyticus) and the rotor protein FliG (7). In E. coli, each motor can be powered by any number between 1 and at least 11 functionally independent stators (8), which exchange with a membrane-bound pool of "spare" stators on a timescale of minutes (9).The most important biophysical method for studying the torque-generating mechanism of the BFM has been to measure its torque-speed curves. This has been done using varying viscous load (10-14) or external torque (15, 16) to control the speed. Fu...

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanism and kinetics of a sodium-driven bacterial flagellar motor

Sowa

Pilizota

et al. 2013

104

“…This indicates there are static interaction potentials between the rotor and de-energized stator units. A cycle that alternates between a set of periodic potentials is a key element in models of the motor mechanism (34,35), raising the possibility that de-energized stator units are locked in one of the motor's working states (25). Periodic, static rotor-stator interactions have been reported before in de-energized motors (36) and in the stop state of the Rhodobacter sphaeroides motor (37).…”

Section: Discussionmentioning

confidence: 99%

Hybrid-fuel bacterial flagellar motors inEscherichia coli

Sowa

Homma

Ishijima

et al. 2014

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H + or Na + ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H + units when Na + is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H + and Na + stator components sharing a common rotor. molecular motor | nano-machine | Na + -driven flagella | hybrid-fuel motor | stator dynamics

“…Deletion of residues 52-65 just after the B segment causes proton leakage and cell growth arrest, which suggests that this fragment acts as a plug to suppress proton leakage (27). The generation of torque is hypothesized to originate from the conformational changes of the MotA cytoplasmic domain upon proton association/dissociation at the carboxyl group of Asp32 on MotB and by the interaction with FliG in the rotor (28)(29)(30)(31).…”

Section: Significancementioning

confidence: 99%

Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor

Nishihara

Kitao

2015

The proton permeation process of the stator complex MotA/B in the flagellar motor of Escherichia coli was investigated. The atomic model structure of the transmembrane part of MotA/B was constructed based on the previously published disulfide cross-linking and tryptophan scanning mutations. The dynamic permeation of hydronium/ sodium ions and water molecule through the channel formed in MotA/B was observed using a steered molecular dynamics simulation. During the simulation, Leu46 of MotB acts as the gate for hydronium ion permeation, which induced the formation of water wire that may mediate the proton transfer to Asp32 on MotB. Free energy profiles for permeation were calculated by umbrella sampling. The free energy barrier for H 3 O + permeation was consistent with the proton transfer rate deduced from the flagellar rotational speed and number of protons per rotation, which suggests that the gating is the rate-limiting step. Structure and dynamics of the MotA/B with nonprotonated and protonated Asp32, Val43Met, and Val43Leu mutants in MotB were investigated using molecular dynamics simulation. A narrowing of the channel was observed in the mutants, which is consistent with the size-dependent ion selectivity. In MotA/B with the nonprotonated Asp32, the A3 segment in MotA maintained a kink whereas the protonation induced 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, which may be correlated to the motion of the flagellar rotor.proton transfer | bacterial flagellar motor | channel gating | ratchet motion | molecular dynamics B acterial flagella are multifuel engines that convert ion motive force to molecular motor rotation. Escherichia coli has a few proton-driven flagellar motors with stators (protein MotA/B complex) in the inner membrane that act as proton channels (1-5). In addition, Vibrio alginolyticus has a polar flagellum powered by sodium ions (6). Bacillus alcalophilus has motors driven by rubidium (Rb + ), potassium (K + ), and sodium ions (Na + ) that can be converted to Na + -driven motors by a single mutation (7).The proton transfer mechanism in membrane proteins is associated with water wire and/or a hydrogen bond chain (HBC) (8, 9). The water wire comprises water molecules aligned in a protein channel, where protons are transferred by hopping along the wire. Protons are conducted through the hydrogen bonds formed by the polar amino acid residues and water molecules along the proton transfer pathway in the HBC. Protons can also be transferred by diffusion of hydronium ions (H 3 O + ). The diffusion distance in a hydrophilic environment is short in a liquid (the lifetime in water is ca. 1 ps) (10, 11), but it should be longer in a more hydrophobic environment. H 3 O + forms a hydrogen bond (H bond) with the nearest neighbor water molecules and the carbonyl groups, and proton hopping along the H bonds is faster than diffusion of Na ...