Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

Vartanian, Armand S.; Paz, Adrian; Fortgang, E.A.; Abramson, Jeff; Dahlquist, Frederick W.

doi:10.1074/jbc.c112.378380

Cited by 62 publications

(98 citation statements)

References 31 publications

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“…FliG N and FliG M are important for flagellar assembly and rotational switching (19), whereas FliG C is primarily responsible for torque generation (20). The atomic structure of FliG has been reported for each domain (21)(22)(23)(24)(25) as well as for the full-length protein (26). Structural analysis showed that FliG C forms a single globular domain with a cluster of charged residues aligned to form a "V"-like ridge on the surface.…”

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confidence: 99%

Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus

et al. 2015

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Rotation of the polar flagellum of Vibrio alginolyticus is driven by a Na؉ -type flagellar motor. FliG, one of the essential rotor proteins located at the upper rim of the C ring, binds to the membrane-embedded MS ring. The MS ring is composed of a single membrane protein, FliF, and serves as a foundation for flagellar assembly. Unexpectedly, about half of the Vibrio FliF protein produced at high levels in Escherichia coli was found in the soluble fraction. Soluble FliF purifies as an oligomer of ϳ700 kDa, as judged by analytical size exclusion chromatography. By using fluorescence correlation spectroscopy, an interaction between a soluble FliF multimer and FliG was detected. This binding was weakened by a series of deletions at the C-terminal end of FliF and was nearly eliminated by a 24-residue deletion or a point mutation at a highly conserved tryptophan residue (W575). Mutations in FliF that caused a defect in FliF-FliG binding abolish flagellation and therefore confer a nonmotile phenotype. As data from in vitro binding assays using the soluble FliF multimer correlate with data from in vivo functional analyses, we conclude that the C-terminal region of the soluble form of FliF retains the ability to bind FliG. Our study confirms that the C-terminal tail of FliF provides the binding site for FliG and is thus required for flagellation in Vibrio, as reported for other species. This is the first report of detection of the FliF-FliG interaction in the Na ؉ -driven flagellar motor, both in vivo and in vitro. Many motile bacteria can swim in liquid environments by means of a motility organelle, the flagellum. Bacteria propel themselves by rotating a helical flagellar filament to move forward, and flagellar rotation is driven by a reversible rotary motor at its base. The flagellum is divided into three parts: the filament (screw), the hook (universal joint), and the basal body (motor). About 50 gene products are required for flagellar assembly and function (1, 2). The energy source for the flagellar motor is the electrochemical gradient of protons or, in some species, sodium ions. The ion flux through stator units that are incorporated around the rotary part of the motor (rotor) is coupled with the generation of torque. The flagellar motor can rotate up to 1,700 revolutions per second (rps) (in the case of the Na ϩ -driven Vibrio motor) and can switch its rotational direction within a millisecond, properties which identify it as an elaborate biological nanomachine (3). However, the key question, how is the rotor-stator interaction coupled to ion flux to generate motor torque, has remained a mystery.Genetic, biochemical, and structural analyses identified key proteins that are most closely involved in torque generation: the stator complex and FliG in the rotor (Fig. 1A) (4). The stator is composed of two membrane proteins (MotA and MotB or their orthologs) that form an ion-conducting force-generating unit (5, 6). In the H ϩ -driven Escherichia coli or Salmonella motor, MotA, with 4 transmembrane (TM) segments, and M...

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Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus

et al. 2015

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“…The crystal structure of the middle domain of FliM (FliMm) reveals a topology similar to CheC (30). The conserved GGXG motif is essential for binding FliG as evident in the FliG⅐FliM complex structure (24,31). The C-terminal domain of FliM interacts with the third rotor protein, FliN (25,27,32).…”

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Structure and Activity of the Flagellar Rotor Protein FliY

Sircar

Greenswag

Bilwes

et al. 2013

Journal of Biological Chemistry

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“…The motor C-rings of these bacteria contain 26 copies of FliG, 34 copies of FliM and >110 copies of FliN (7)(8)(9). Most of these structures were determined using proteins from other organisms, and their assembly models have been recently proposed (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Electron microscopic and tomographic studies have shown that the organization of the switch proteins is similar across different species such that FliG is the closest to the cytoplasmic membrane, followed by FliM and FliN is found in a distant membrane location, toward the cytoplasm ( Figure 1A) (4,23,24).…”

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Three SpoA-domain proteins interact in the creation of the flagellar type III secretion system in Helicobacter pylori

Lam

Xue

Sun

et al. 2018

Journal of Biological Chemistry

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Bacterial flagella are rotary nano-machines that contribute to bacterial fitness in many settings, including host colonization. The flagellar motor relies on the multiprotein flagellar motor-switch complex to govern flagellum formation and rotational direction. Different bacteria exhibit great diversity in their flagellar motors. One such variation is exemplified by the motor-switch apparatus of the gastric pathogen Helicobacter pylori, which carries an extra switch protein, FliY, along with the more typical FliG, FliM, and FliN proteins. All switch proteins are needed for normal flagellation and motility in H. pylori, but the molecular mechanism of their assembly is unknown. To fill this gap, we examined the interactions among these proteins. We found that the C-terminal SpoA domain of FliY (FliY C ) is critical to flagellation and forms heterodimeric complexes with the FliN and FliM SpoA domains, which are β-sheet domains of type III secretion system proteins. Surprisingly, unlike in other flagellar switch systems, neither FliY nor FliN self-associated. The crystal structure of the FliY C -FliN C complex revealed a saddle-shape structure homologous to the FliN-FliN dimer of Thermotoga maritima, consistent with a FliY-FliN heterodimer forming the functional unit. Analysis of the FliY C -FliN C interface indicated that oppositely charged residues specific to each protein drive heterodimer formation. Moreover, both FliY C -FliM C and FliY C -FliN C associated with the flagellar regulatory protein FliH, explaining their important roles in flagellation. We conclude that H. pylori uses a FliY-FliN heterodimer instead of a homodimer and creates a switch complex with SpoA domains derived from three distinct proteins.Bacterial flagella are rotary nano-machines that contribute to bacterial fitness in a variety of settings, including mammalian and plant colonization (1,2). Although the basic function of flagella as a motor organelle is conserved, substantial variation exists among microbes in the components used to build and operate key aspects of the flagella. For example, we now know that there are diverse motor structures from cryo-electron tomography studies (3,4), and that bacterial motors consist of FliG, FliM and either FliN or FliY or the combination of both FliN and FliY (5) The flagellar motor switch complex, also called the C-ring, is found at the base of each flagellum and resides within the cytoplasm (6). It plays an important role in flagellar assembly, torque generation, and rotational switching. Numerous studies have dissected the composition, arrangement, and structure of the switch proteins, with a focus on those from Escherichia coli and Salmonella typhimurium that possess FliG, FliM and FliN. The motor C-rings of these bacteria contain 26 copies of FliG, 34 copies of FliM and >110 copies of . Most of these structures were determined using proteins from other organisms, and their assembly models have been recently proposed (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Electron microscop...

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Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

Cited by 62 publications

References 31 publications

Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus

Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus

Structure and Activity of the Flagellar Rotor Protein FliY

Three SpoA-domain proteins interact in the creation of the flagellar type III secretion system in Helicobacter pylori

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Structure of Flagellar Motor Proteins in Complex Allows for Insights into Motor Structure and Switching

Cited by 62 publications

References 31 publications

Interaction of the C-Terminal Tail of FliF with FliG from the Na + -Driven Flagellar Motor of Vibrio alginolyticus

Interaction of the C-Terminal Tail of FliF with FliG from the Na + -Driven Flagellar Motor of Vibrio alginolyticus

Structure and Activity of the Flagellar Rotor Protein FliY

Three SpoA-domain proteins interact in the creation of the flagellar type III secretion system in Helicobacter pylori

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Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus

Interaction of the C-Terminal Tail of FliF with FliG from the Na ⁺ -Driven Flagellar Motor of Vibrio alginolyticus