Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

Zhu, S. J.; Takao, M.; Li, Na; Sakuma, Mayuko; Nishino, Yuuki; Homma, Michio; Kojima, Seiji; Imada, Katsumi

doi:10.1073/pnas.1324201111

Cited by 87 publications

(117 citation statements)

References 36 publications

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“…For V. fischeri, these proposed stator complexes were consistent with the size of a recent single-particle reconstruction of the Vibrio stator complex (16). The densities spanned the C-ring to ∼10 nm above the inner membrane, as previously predicted (34), were arranged in a 17-nm radius ring with a with 13-fold rotational symmetry, and contacted the 16-nm radius T-ring ( Fig. 2 A-D, red arrows; continuous density is best visualized in Fig.…”

Section: Three Flagellar Motors That Produce Different Torques Are Stsupporting

confidence: 88%

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Beeby

Ribardo

Brennan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

185

262

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Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

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Section: Three Flagellar Motors That Produce Different Torques Are Stsupporting

confidence: 88%

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Beeby

Ribardo

Brennan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

185

262

View full text Add to dashboard Cite

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“…Considering the torque produced by N stators as Nτ 1 = γ2πω N (where τ 1 is the torque produced by a single stator, γ the drag coefficient of the bead, and ω N the measured speed (in Hz) of the motor with N stators), all the strains tested are driven by N ~ 8-10 stators, in line with previous measurements at high load 15,44 . This result supports previous works focused on quantifying steady-state stator stoichiometry by fluorescent stators 15,[18][19][20][21]28,29,[45][46][47] .…”

Section: Resultssupporting

confidence: 91%

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

Heo

Nord

Chamousset

et al. 2018

Biophysical Journal

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Fluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects, but such insight is still lacking for many applications. This is particularly relevant in the study of the internal dynamics of motor proteins, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to the stator of the Bacterial Flagellar Motor (BFM) have previously been used to unveil the motor subunit dynamics. Here we report the effects on single motors of three fluorescent proteins fused to the stators, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry remained unaffected. MotB fusions decreased the switching frequency and induced a novel bias-dependent asymmetry in the speed in the two directions. These effects could be mitigated by inserting a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions to the stator on BFM dynamics and their alleviation-new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Fusion of a fluorescent protein to a protein of interest is an invaluable tool for the study of protein function in a broad, and still expanding range of in vivo and in vitro systems. However, it is widely recognized that the presence of fluorescent proteins can alter functional properties of the native protein [1][2][3][4][5] . Careful assessment of these side effects and strategies to minimize them have therefore been critical to the success of fluorescent protein fusions (FPFs) [6][7][8][9] . The continuing development of FPF-based approaches to explore novel phenomena also calls for new insight into the associated side effects and methods to alleviate them [10][11][12] . One direction of research for which this holds is the study of the internal dynamics of proteins complexes 13,14 . Recently, several studies have made use of FPFs to study the internal subunit dynamics of the bacterial flagellar motor (BFM) [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29] . Although FPFs were shown to affect BFM function by decreasing the chemotactic motility of cells and average speed of motors 15 , a quantitative characterization of the impact on the BFM mechanical behavior is lacking. Such perturbations limit the depth to which the internal dynamics and function of the BFM, and protein complexes in general, can be probed with FPFs. Linker peptides inserted at the fusion point have been instrumental in minimizing non-native behaviour of FPFs in many biological systems [8][9][10][11] , but their use in the study of protein-complex dynamics is limited in the BFM has thus far been limited.The BFM is located at the base of each flagellum in the membrane of many motile bacteria (Fig. 1A). The torque gene...

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“…It has been suggested that force upon the PG may cause structural changes in stator binding sites which may affect stator unit association rate (17). Additionally, crystal structures of MotBC of Salmonella and PomBC of Vibrio suggest that a drastic conformational change in the N-terminal portion of MotBC /PomBC is required for the stator to bind to the PG (38,39). Mutational studies suggest that this conformational change may be triggered by an interaction between the cytoplasmic domain of MotA and FliG (40), a process potentially complicated by the rotation of the rotor.…”

Section: Discussionmentioning

confidence: 99%

“…However, interestingly, it has been reported that the putative key PGB residues in the structure of the PGB dimer of MotB are buried and not readily accessible (44) and that a substantial structural flexibility of the domain is considered necessary to mask and unmask them (45). The Nterminal portion of MotB must perform a large conformational extension to reach the PG (38,39). Recently, a conformational change upon binding has also been hypothesized in the PGassociated C-terminal of the closely related Outer Membrane protein OmpA (46).…”

Section: Discussionmentioning

confidence: 99%

A Catch-Bond Drives Stator Mechanosensitivity in the Bacterial Flagellar Motor

Nord

Gachon

Pérez-Carrasco

et al. 2018

Biophysical Journal

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The bacterial flagellar motor (BFM) is the rotary motor that rotates each bacterial flagellum, powering the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force-powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechanosensitive, with the number of engaged units dependent on the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechanosensitivity is unknown. Here, we directly measure the kinetics of arrival and departure of the stator units in individual motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. The kinetic rates obtained, robust with respect to the details of the applied adsorption model, indicate that the lifetime of an assembled stator unit increases when a higher force is applied to its anchoring point in the cell wall. This provides strong evidence that a catch bond (a bond strengthened instead of weakened by force) drives mechanosensitivity of the flagellar motor complex. These results add the BFM to a short, but growing, list of systems demonstrating catch bonds, suggesting that this "molecular strategy" is a widespread mechanism to sense and respond to mechanical stress. We propose that force-enhanced stator adhesion allows the cell to adapt to a heterogeneous environmental viscosity and may ultimately play a role in surface-sensing during swarming and biofilm formation.bacterial flagellar motor | molecular motor | mechanosensitivity | catch bond | Escherichia coli T he bacterial flagellar motor (BFM) is a large molecular complex found in many species of motile bacteria which actively rotates each flagellum of the cell, enabling swimming, chemotaxis, and swarming (1, 2). The rotor of the BFM is embedded within and spans the cellular membranes, coupling rotation to the extracellular hook and flagellar filament. Multiple transmembrane complexes, called stator units, are anchored around the perimeter of the rotor and bound to the rigid peptidoglycan (PG) layer (3-5). By harnessing the ion motive force, the stator units are responsible for torque generation, acting upon the common ring formed by FliG proteins on the cytosolic side of the rotor (Fig. 1A) (6, 7).Several studies have revealed the continuous exchange of various BFM molecular constituents (8-10), demonstrating that a static model for the BFM structure is not adequate. A prime example, and in contrast to macroscopic rotary motors, the stator of the BFM is dynamic; while each bound stator unit acts upon the rotor independently (11, 12), their stoichiometry in the motor varies. Once anchored around the rotor, stator units dynamically turn over, eventually diffusing away in the inner membrane (10). Each additional recruited stator unit increases the total torque and thus the measured rotational speed of the motor (11, 13), and up to 11 units have been observed to engage in an individual motor in Esche...

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Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor

Cited by 87 publications

References 36 publications

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Impact of Fluorescent Protein Fusions on the Bacterial Flagellar Motor

A Catch-Bond Drives Stator Mechanosensitivity in the Bacterial Flagellar Motor

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