Dataset Information

Cooperative stator assembly of bacterial flagellar motor mediated by rotation.

ABSTRACT: Cooperativity has a central place in biological regulation, providing robust and highly-sensitive regulation. The bacterial flagellar motor implements autonomous torque regulation based on the stator's dynamic structure; the stator units bind to and dissociate from the motor dynamically in response to environmental changes. However, the mechanism of this dynamic assembly is not fully understood. Here, we demonstrate the cooperativity in the stator assembly dynamics. The binding is slow at the stalled state, but externally forced rotation as well as driving by motor torque in either direction boosts the stator binding. Hence, once a stator unit binds, it drives the rotor and triggers the avalanche of succeeding bindings. This cooperative mechanism based on nonequilibrium allostery accords with the recently-proposed gear-type coupling between the rotor and stator.

SUBMITTER: Ito KI

PROVIDER: S-EPMC8163892 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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Cooperative stator assembly of bacterial flagellar motor mediated by rotation.

Ito Kenta I KI Nakamura Shuichi S Toyabe Shoichi S

Nature communications 20210528 1

Cooperativity has a central place in biological regulation, providing robust and highly-sensitive regulation. The bacterial flagellar motor implements autonomous torque regulation based on the stator's dynamic structure; the stator units bind to and dissociate from the motor dynamically in response to environmental changes. However, the mechanism of this dynamic assembly is not fully understood. Here, we demonstrate the cooperativity in the stator assembly dynamics. The binding is slow at the st ...[more]

PMID: 34050167

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Project description:The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses, but have not been demonstrated biochemically. Here, we used site-directed photo- and disulfide-crosslinking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-L-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA formed a crosslink with FliG. PomA residue K89 gave the highest yield of crosslinks, suggesting that it is the PomA residue nearest to FliG. UV-induced crosslinking stopped motor rotation, and the isolated hook-basal body contained the crosslinked products. pBPA inserted to replace residues R281 or D288 in FliG formed crosslinks with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide crosslinks with cysteine inserted in place of FliG residues R281 and D288, and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA.ImportanceThe bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo- and disulfide-crosslinking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

Project description:Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extend from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA (similar to MotA in Escherichia coli) and PomB (similar to MotB in E. coli), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-cross-linking and disulfide cross-linking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after cross-linking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. IMPORTANCE The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by cross-linking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.

Dataset Information

Cooperative stator assembly of bacterial flagellar motor mediated by rotation.

Publications

Cooperative stator assembly of bacterial flagellar motor mediated by rotation.

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