Molecular mechanism for rotational switching of the bacterial flagellar motor.
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ABSTRACT:
SUBMITTER: Chang Y
PROVIDER: S-EPMC8129871 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The bacterial flagellar motor can rotate either clockwise (CW) or counterclockwise (CCW). Three flagellar proteins, FliG, FliM, and FliN, are required for rapid switching between the CW and CCW directions. Switching is achieved by a conformational change in FliG induced by the binding of a chemotaxis signaling protein, phospho-CheY, to FliM and FliN. FliG consists of three domains, FliG(N), FliG(M), and FliG(C), and forms a ring on the cytoplasmic face of the MS ring of the flagellar basal body. Crystal structures have been reported for the FliG(MC) domains of Thermotoga maritima, which consist of the FliG(M) and FliG(C) domains and a helix E that connects these two domains, and full-length FliG of Aquifex aeolicus. However, the basis for the switching mechanism is based only on previously obtained genetic data and is hence rather indirect. We characterized a CW-biased mutant (fliG(?PAA)) of Salmonella enterica by direct observation of rotation of a single motor at high temporal and spatial resolution. We also determined the crystal structure of the FliG(MC) domains of an equivalent deletion mutant variant of T. maritima (fliG(?PEV)). The FliG(?PAA) motor produced torque at wild-type levels under a wide range of external load conditions. The wild-type motors rotated exclusively in the CCW direction under our experimental conditions, whereas the mutant motors rotated only in the CW direction. This result suggests that wild-type FliG is more stable in the CCW state than in the CW state, whereas FliG(?PAA) is more stable in the CW state than in the CCW state. The structure of the TM-FliG(MC)(?PEV) revealed that extremely CW-biased rotation was caused by a conformational change in helix E. Although the arrangement of FliG(C) relative to FliG(M) in a single molecule was different among the three crystals, a conserved FliG(M)-FliG(C) unit was observed in all three of them. We suggest that the conserved FliG(M)-FliG(C) unit is the basic functional element in the rotor ring and that the PAA deletion induces a conformational change in a hinge-loop between FliG(M) and helix E to achieve the CW state of the FliG ring. We also propose a novel model for the arrangement of FliG subunits within the motor. The model is in agreement with the previous mutational and cross-linking experiments and explains the cooperative switching mechanism of the flagellar motor.
| S-EPMC3091841 | biostudies-literature
Project description:The high-resolution structures of nearly all the proteins that comprise the bacterial flagellar motor switch complex have been solved; yet a clear picture of the switching mechanism has not emerged. Here, we used NMR to characterize the interaction modes and solution properties of a number of these proteins, including several soluble fragments of the flagellar motor proteins FliM and FliG, and the response-regulator CheY. We find that activated CheY, the switch signal, binds to a previously unidentified region of FliM, adjacent to the FliM-FliM interface. We also find that activated CheY and FliG bind with mutual exclusivity to this site on FliM, because their respective binding surfaces partially overlap. These data support a model of CheY-driven motor switching wherein the binding of activated CheY to FliM displaces the carboxy-terminal domain of FliG (FliGC) from FliM, modulating the FliGC-MotA interaction, and causing the motor to switch rotational sense as required for chemotaxis.
| S-EPMC3327947 | biostudies-literature
Project description:Bacteria sense temporal changes in extracellular stimuli via sensory signal transducers and move by rotating flagella towards into a favorable environment for their survival. 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 a 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. 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. In this mini-review, we will describe current understanding of the switching mechanism of the bacterial flagellar motor.
| S-EPMC6700473 | biostudies-literature
Project description:The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.
| S-EPMC3159035 | biostudies-literature
Project description:Many bacteria are propelled by flagellar motors that stochastically switch between the clockwise and counterclockwise rotation direction. Although the switching dynamics is one of their most important characteristics, the mechanisms that control it are poorly understood. We present a statistical-mechanical model of the bacterial flagellar motor. At its heart is the assumption that the rotor protein complex, which is connected to the flagellum, can exist in two conformational states and that switching between these states depends on the interactions with the stator proteins, which drive the rotor. This couples switching to rotation, making the switch sensitive to torque and speed. Another key element is that after a switch, it takes time for the load to build up, due to conformational transitions of the flagellum. This slow relaxation dynamics of the filament leads, in combination with the load dependence of the switching frequency, to a characteristic switching time, as recently observed. Hence, our model predicts that the switching dynamics is not only controlled by the chemotaxis-signaling network, but also by mechanical feedback of the flagellum.
| S-EPMC2779088 | biostudies-other
Project description:The direction of flagellar rotation is regulated by a rotor-mounted protein assembly, termed the "switch complex," formed from multiple copies of the proteins FliG, FliM, and FliN. The structures of major parts of these proteins are known, and the overall organization of proteins in the complex has been elucidated previously using a combination of protein-binding, mutational, and cross-linking approaches. In Escherichia coli, the switch from counterclockwise to clockwise rotation is triggered by the signaling protein phospho-CheY, which binds to the lower part of the switch complex and induces small movements of FliM and FliN subunits relative to each other. Direction switching also must produce movements in the upper part of the complex, particularly in the C-terminal domain of FliG (FliG(C)), which interacts with the stator to generate the torque for flagellar rotation. In the present study, protein movements in the middle and upper parts of the switch complex have been probed by means of targeted cross-linking and mutational analysis. Switching induces a tilting movement of the FliM domains that form the middle part of the switch and a consequent rotation of the affixed FliG(C) domains that reorients the stator interaction sites by about 90°. In a recently proposed hypothesis for the motor mechanism, such a reorientation of FliG(C) would reverse the direction of motor rotation.
| S-EPMC3193218 | biostudies-literature
Project description:Switching dynamics of flagellar motors of Escherichia coli is commonly observed through markers attached to the flagellar filaments. To eliminate possible complications resulting from the conformational transitions of these filaments and to look at the output of motors more directly, we monitored motor rotation by attaching nanogold spheres to the hooks of cells lacking filaments. We observed exponentially distributed counterclockwise (CCW) and clockwise (CW) intervals and Lorentzian power spectra of the switching time series consistent with models that treat motor switching as a two-state Poisson process.
| S-EPMC4226075 | biostudies-literature
Project description:The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.
| S-EPMC7505661 | biostudies-literature
Project description:The bacterial flagellar motor plays a crucial role in both bacterial locomotion and chemotaxis. Recent experiments reveal that the switching dynamics of the motor depend on the rotation speed of the motor, and thus the motor torque, nonmonotonically. Here we present a unified mathematical model which treats motor torque generation based on experimental torque-speed curves and the torque-dependent switching based on the conformational spread model. The model successfully reproduces the observed switching rate as a function of the rotation speed, and provides a generic physical explanation independent of most details. A stator affects the switching dynamics through two mechanisms: accelerating the conformational flipping rate of individual rotor-switching units, which contributes most when the stator works at a high torque and thus a low speed; and influencing a larger number of rotor-switching units within unit time, whose contribution is the greatest when the motor rotates at a high speed. Consequently, the switching rate shows a maximum at intermediate speed, where the above two mechanisms find an optimal output. The load-switching relation may serve as a mechanism for sensing the physical environment, similar to the chemotaxis mechanism for sensing the chemical environment. It may also coordinate the switch dynamics of motors within the same cell.
| S-EPMC3558881 | biostudies-literature
Project description: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.
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