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Rotational Mechanism Model of the Bacterial V₁ Motor Based on Structural and Computational Analyses.

ABSTRACT: V₁-ATPase exemplifies the ubiquitous rotary motor, in which a central shaft DF complex rotates inside a hexagonally arranged catalytic A₃B₃ complex, powered by the energy from ATP hydrolysis. We have recently reported a number of crystal structures of the Enterococcus hirae A₃B₃DF (V₁) complex corresponding to its nucleotide-bound intermediate states, namely the forms waiting for ATP hydrolysis (denoted as catalytic dwell), ATP binding (ATP-binding dwell), and ADP release (ADP-release dwell) along the rotatory catalytic cycle of ATPase. Furthermore, we have performed microsecond-scale molecular dynamics simulations and free-energy calculations to investigate the conformational transitions between these intermediate states and to probe the long-time dynamics of the molecular motor. In this article, the molecular structure and dynamics of the V₁-ATPase are reviewed to bring forth a unified model of the motor's remarkable rotational mechanism.

SUBMITTER: Singharoy A

PROVIDER: S-EPMC6371843 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Rotational Mechanism Model of the Bacterial V1 Motor Based on Structural and Computational Analyses.

Singharoy Abhishek A Chipot Chris C Ekimoto Toru T Suzuki Kano K Ikeguchi Mitsunori M Yamato Ichiro I Murata Takeshi T

Frontiers in physiology 20190205

V1-ATPase exemplifies the ubiquitous rotary motor, in which a central shaft DF complex rotates inside a hexagonally arranged catalytic A3B3 complex, powered by the energy from ATP hydrolysis. We have recently reported a number of crystal structures of the Enterococcus hirae A3B3DF (V1) complex corresponding to its nucleotide-bound intermediate states, namely the forms waiting for ATP hydrolysis (denoted as catalytic dwell), ...[more]

PMID: 30804798

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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.

Project description:Survival of species in an ever-changing environment requires a flexibility that extends beyond merely selecting the most appropriate actions. It also involves readiness to stop or switch actions in response to environmental changes. Although considerable research has been devoted to understanding how the brain switches actions, the computations underlying the switching process and how it relates to the selecting and stopping processes remain elusive. A normative theory suggests that switching is simply an extension of the stopping process, during which a current action is first inhibited by an independent pause mechanism before a new action is generated. This theory was challenged by the affordance competition hypothesis, according to which the switching process is implemented through a competition between the current and new actions, without engaging an independent pause mechanism. To delineate the computations underlying these action regulation functions, we utilized a neurocomputational theory that models the process of selecting, stopping and switching reaching movements. We tested the model predictions in healthy individuals who performed reaches in dynamic and uncertain environments that often required stopping and switching actions. Our findings suggest that unlike the stopping process, switching does not necessitate a proactive pause mechanism to delay movement initiation. Hence, the switching and stopping processes seem to be implemented by different mechanisms at the planning phase of the reaching movement. However, once the reaching movement has been initiated, the switching process seems to involve an independent pause mechanism if the new target location is unknown prior to movement initiation. These findings offer a new understanding of the computations underlying action switching, contribute valuable insights into the fundamental neuroscientific mechanisms of action regulation, and open new avenues for future neurophysiological investigations.

Dataset Information

Rotational Mechanism Model of the Bacterial V₁ Motor Based on Structural and Computational Analyses.

Publications

Rotational Mechanism Model of the Bacterial V<sub>1</sub> Motor Based on Structural and Computational Analyses.

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