Multiple Conformations of the FliG C-Terminal Domain Provide Insight into Flagellar Motor Switching

Lam, Kwok Ho; Ip, Wing-Sang; Lam, Yun Wah; Chan, Sun On; Ling, T. K. W.; Au, Shannon Wing-Ngor

doi:10.1016/j.str.2011.11.020

Cited by 40 publications

(70 citation statements)

References 53 publications

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“…We initially attempted molecular replacement with A. aeolicus FliG as a template model, but no solution could be found, indicating that the two FliG N sstructures were different. Interestingly, the top hit (z-score 7.4) from a search for structural homology using Dali (16) revealed the ARM motif of the FliG middle domain from A. aeolicus (13) and H. pylori (15). The structural alignment of helices 2-5 from H. pylori FliG N with the ARM M gave a root-mean-square deviation (RMSD) Cα of 1.9 Å for (A. aeolicus) and 2.1 Å for (H. pylori) (Supplemental Figure 3).…”

Section: Resultsmentioning

confidence: 99%

“…The percentage of flagellated cells was further reduced in the V34D/L81D mutant strain, suggesting that the hydrophobic surfaces of helices 4 and 6 were essential for proper flagellar assembly. To further clarify the effect of these mutations on motor function, we studied the swimming behavior by fixed-time diffusion analysis (15,19). This method measures how close the swimming behavior of a bacterium is to pure diffusion.…”

Section: Downloaded Frommentioning

confidence: 99%

“…Although tremendous effort has been spent on understanding the FliG structure in switch assembly, torque generation and rotational switching, the atomic details of the initial phase of the MS-C ring complex formation remain poorly understood. The crystal structures of FliG from various bacterial species have been determined (13)(14)(15). FliG is comprised of three domains: FliG N , FliG M , and FliG C , where the subscripts denote the N-terminal, middle and C-terminal domains, respectively.…”

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Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Xue

Lam

Zhang

et al. 2018

Journal of Biological Chemistry

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The bacterial flagellar motor is a self-assembling supramolecular nanodevice.Its spontaneous biosynthesis is initiated by the insertion of the MS ring protein FliF into the inner membrane, followed by attachment of the switch protein FliG. Assembly of this multiprotein complex is tightly regulated to avoid nonspecific aggregation, but the molecular mechanisms governing flagellar assembly are unclear. Here, we present the crystal structure of the cytoplasmic domain of FliF complexed with the Nterminal domain of FliG (FliF C -FliG N ) from the bacterium Helicobacter pylori. Within this complex, FliF C interacted with FliG N through extensive hydrophobic contacts similar to those observed in the FliF C -FliG N structure from the thermophile Thermotoga maritima, indicating conservation of the FliF C -FliG N interaction across bacterial species. Analysis of the crystal lattice revealed that the heterodimeric complex packs as a linear superhelix via stacking of the armadillo-repeat-like motifs (ARM) of FliG N . Notably, this linear helix was similar to that observed for the assembly of the FliG middle domain. We validated the in vivo relevance of the FliG N stacking by complementation studies in Escherichia coli. Furthermore, structural comparison with apo FliG from the thermophile Aquifex aeolicus indicated that FliF regulates the conformational transition of FliG and exposes the complementary ARM-like motifs of FliG N , containing conserved hydrophobic residues. FliF apparently both provides a template for FliG polymerization and spatiotemporally controls subunit interactions within FliG. Our findings reveal that a small protein fold can serve as a versatile building block to assemble into a multiprotein machinery of distinct shapes for specific functions.The bacterial flagellar motor is a self-assembled reversible rotary nanodevice. This dynamic rotary motor of about 40 nm in diameter is composed of rings of protein oligomers: the L ring (outer membrane), P ring (peptidoglycan layer), MS ring (inner membrane) and C ring (cytoplasm) (1,2). The L and P rings are believed to act as a bushing through which a central rotating rod can pass. The MS and C rings contribute to the rotor part of the flagellar motor. Torque is generated by a membrane-bound stator, which converts electrochemical potential into mechanical force that acts on the rotor. The synthesis of the flagellum begins at the rotor, which is composed of the MS and C ring protein subassemblies ( Figure 1). Specifically, the assembly of the MS ring protein FliF prompts the incorporation of the switch protein FliG and subsequently the proteins FliM and FliN to form the cytoplasmic motor switch complex. The MS ring plays an important role as it interacts both with the rod where the flagellum is anchored and with the C ring where torque Crystal structure of FliF-FliG complex from H. pylori generation and rotation switching take place.In Salmonella typhimurium and Escherichia coli, there are about 26 copies of FliF in the MS ring and 26 copies of FliG, 34 c...

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Section: Resultsmentioning

confidence: 99%

Section: Downloaded Frommentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Xue

Lam

Zhang

et al. 2018

Journal of Biological Chemistry

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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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“…Some point mutations or deletions of a few specific residues in FliG lock motor rotation in either the CCW or CW direction (47)(48)(49). Several switching models have recently been reported based on structural differences between a CW-locked FliG mutant protein and wild-type FliG (15,43,49,50).…”

Section: Discussionmentioning

confidence: 99%

High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor

et al. 2013

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hThe bacterial flagellar motor is a reversible rotary machine that rotates a left-handed helical filament, allowing bacteria to swim toward a more favorable environment. The direction of rotation reverses from counterclockwise (CCW) to clockwise (CW), and vice versa, in response to input from the chemotaxis signaling circuit. CW rotation is normally caused by binding of the phosphorylated response regulator CheY (CheY-P), and strains lacking CheY are typically locked in CCW rotation. The detailed mechanism of switching remains unresolved because it is technically difficult to regulate the level of CheY-P within the concentration range that produces flagellar reversals. Here, we demonstrate that high hydrostatic pressure can induce CW rotation even in the absence of CheY-P. The rotation of single flagellar motors in Escherichia coli cells with the cheY gene deleted was monitored at various pressures and temperatures. Application of >120 MPa pressure induced a reversal from CCW to CW at 20°C, although at that temperature, no motor rotated CW at ambient pressure (0.1 MPa). At lower temperatures, pressure-induced changes in direction were observed at pressures of <120 MPa. CW rotation increased with pressure in a sigmoidal fashion, as it does in response to increasing concentrations of CheY-P. Application of pressure generally promotes the formation of clusters of ordered water molecules on the surfaces of proteins. It is possible that hydration of the switch complex at high pressure induces structural changes similar to those caused by the binding of CheY-P. Escherichia coli cells can sense and respond to many environmental factors, such as chemicals, pH, and temperature, and swim toward more favorable environments for them by rotating their helical flagella (1-5). Each flagellum includes a long (ϳ10-m), thin (ϳ20-nm), helical filament that turns about its long axis either counterclockwise (CCW) (viewed from filament to motor) or clockwise (CW). CCW rotation allows the multiple filaments of a cell to form a helical bundle that propels the cell body smoothly in what is called a "run." A CW-rotating filament leaves the bundle and leads to a change in the swimming direction, called a "tumble." Regulating switching between CCW and CW rotation leads E. coli cells to bias a three-dimensional random walk to directional movements in liquid environments.The bacterial flagellar motor converts the chemical energy of ion flux across the cell membrane to the rotation of a flagellum (6-10). The motor consists of a rotor and multiple stator units, and its major components are located in the cell membrane. The rotor spins relative to the cell body, and it is firmly connected to the flagellar filament, whereas the stator units are anchored to the cell wall. Torque is generated by intermolecular interactions between a rotor and stator units. The rotational direction is controlled by the binding of the phosphorylated form of the response regulator CheY (CheY-P) onto the rotor. The direction of flagellar rotation is highly dependent o...

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Multiple Conformations of the FliG C-Terminal Domain Provide Insight into Flagellar Motor Switching

Cited by 40 publications

References 53 publications

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

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

High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor

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Multiple Conformations of the FliG C-Terminal Domain Provide Insight into Flagellar Motor Switching

Cited by 40 publications

References 53 publications

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

Crystal structure of the FliF–FliG complex from Helicobacter pylori yields insight into the assembly of the motor MS–C ring in the bacterial flagellum

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

High Hydrostatic Pressure Induces Counterclockwise to Clockwise Reversals of the Escherichia coli Flagellar Motor

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