scholarly journals The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Brittany L Carroll ◽  
Tatsuro Nishikino ◽  
Wangbiao Guo ◽  
Shiwei Zhu ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Electron Tomography ◽  
Protein Composition ◽  
Vibrio Alginolyticus ◽  
Flagellar Motor ◽  
Conformational Rearrangement ◽  
Structural Remodeling ◽  
Cryo Electron Tomography ◽  
Bacterial Flagellar Motor ◽  
Rotational Direction

The bacterial flagellar motor switches rotational direction between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants in Vibrio alginolyticus. We determined the C-ring molecular architectures, providing novel insights into the mechanism of rotational switching. We report that the C-ring maintained 34-fold symmetry in both rotational senses, and the protein composition remained constant. The two structures show FliG conformational changes elicit a large conformational rearrangement of the rotor complex that coincides with rotational switching of the flagellum. FliM and FliN form a stable spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

scholarly journals The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching

2020 ◽  
Author(s):  
Brittany L. Carroll ◽  
Tatsuro Nishikino ◽  
Wangbiao Guo ◽  
Shiwei Zhu ◽  
Seiji Kojima ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Direct Evidence ◽  
Electron Tomography ◽  
Vibrio Alginolyticus ◽  
Flagellar Motor ◽  
Large Rearrangement ◽  
Structural Remodeling ◽  
Cryo Electron Tomography ◽  
Ring Structures ◽  
Bacterial Flagellar Motor

ABSTRACTThe bacterial flagellar motor is an intricate nanomachine that switches rotational directions between counterclockwise (CCW) and clockwise (CW) to direct the migration of the cell. The cytoplasmic ring (C-ring) of the motor, which is composed of FliG, FliM, and FliN, is known for controlling the rotational sense of the flagellum. However, the mechanism underlying rotational switching remains elusive. Here, we deployed cryo-electron tomography to visualize the C-ring in two rotational biased mutants (CCW-biased fliG-G214S and CW-locked fliG-G215A) in Vibrio alginolyticus. Sub-tomogram averaging was utilized to resolve two distinct conformations of the C-ring. Comparison of the C-ring structures in two rotational senses provide direct evidence that the C-ring undergoes major structural remodeling during rotational switch. Specifically, FliG conformational changes elicit a large rearrangement of the C-ring that coincides with rotational switching, whereas FliM and FliN form a spiral-shaped base of the C-ring, likely stabilizing the C-ring during the conformational remodeling.

scholarly journals Bacterial flagellar motor PL-ring disassembly subcomplexes are widespread and ancient

2020 ◽  
Vol 117 (16) ◽  
pp. 8941-8947 ◽  
Author(s):  
Mohammed Kaplan ◽  
Michael J. Sweredoski ◽  
João P. G. L. M. Rodrigues ◽  
Elitza I. Tocheva ◽  
Yi-Wei Chang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Complex Structures ◽  
Flagellar Motor ◽  
Additional Species ◽  
Bacterial Flagellum ◽  
Cryo Electron Tomography ◽  
Show Evidence ◽  
Bacterial Flagellar Motor ◽  
Horizontal Transfers

The bacterial flagellum is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic subcomplex comprising the decorated P and L rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here, we show that this subcomplex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this subcomplex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P and L rings is to seal the outer membrane after motor disassembly.

scholarly journals Molecular Mechanism for Rotational Switching of the Bacterial Flagellar Motor

2020 ◽  
Author(s):  
Yunjie Chang ◽  
Kai Zhang ◽  
Brittany L. Carroll ◽  
Xiaowei Zhao ◽  
Nyles W. Charon ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Conformational Changes ◽  
Proton Motive Force ◽  
Model System ◽  
Dependent Manner ◽  
Flagellar Motor ◽  
Bacterial Flagellar Motor ◽  
Rotational Direction ◽  
Cytoplasmic Side

AbstractThe bacterial flagellar motor is a remarkable nanomachine that can rapidly rotate in both counter-clockwise (CCW) and clockwise (CW) senses. The transitions between CCW and CW rotation are critical for chemotaxis, and they are controlled by a signaling protein (CheY-P) that interacts with a switch complex at the cytoplasmic side of the flagellar motor. However, the exact molecular mechanism by which CheY-P controls the motor rotational switch remains enigmatic. Here, we use the Lyme disease spirochete, Borrelia burgdorferi, as the model system to dissect the mechanism underlying flagellar rotational switching. We first determined high resolution in situ motor structures in the cheX and cheY3 mutants in which motors are genetically locked in CCW or CW rotation. The structures showed that the CheY3 protein of B. burgdorferi interacts directly with the FliM protein of the switch complex in a phosphorylation-dependent manner. The binding of CheY3-P to FliM induces a major remodeling of the switch protein FliG2 that alters its interaction with the torque generator. Because the remodeling of FliG2 is directly correlated with the rotational direction, our data lead to a model for flagellar function in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force. Rapid conformational changes of FliG2 allow the switch complex to interact with opposite sides of the rotating torque generator, thereby facilitating rotational switching between CW and CCW.

scholarly journals Bacterial flagellar motor PL-ring disassembly Sub-complexes are widespread and ancient

2019 ◽  
Author(s):  
Mohammed Kaplan ◽  
Michael J. Sweredoski ◽  
João P.G.L.M. Rodrigues ◽  
Elitza I. Tocheva ◽  
Yi-Wei Chang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Electron Tomography ◽  
Complex Structures ◽  
Flagellar Motor ◽  
Additional Species ◽  
Cellular Evolution ◽  
Cryo Electron Tomography ◽  
Show Evidence ◽  
Bacterial Flagellar Motor ◽  
Horizontal Transfers

AbstractThe bacterial flagellar motor is an amazing nanomachine. Understanding how such complex structures arose is crucial to our understanding of cellular evolution. We and others recently reported that in several Gammaproteobacterial species, a relic sub-complex comprising the decorated P- and L-rings persists in the outer membrane after flagellum disassembly. Imaging nine additional species with cryo-electron tomography, here we show that this sub-complex persists after flagellum disassembly in other phyla as well. Bioinformatic analyses fail to show evidence of any recent horizontal transfers of the P- and L-ring genes, suggesting that this sub-complex and its persistence is an ancient and conserved feature of the flagellar motor. We hypothesize that one function of the P- and L-rings is to seal the outer membrane after motor disassembly.

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

scholarly journals Intact Flagellar Motor of Borrelia burgdorferi Revealed by Cryo-Electron Tomography: Evidence for Stator Ring Curvature and Rotor/C-Ring Assembly Flexion

2009 ◽  
Vol 191 (16) ◽  
pp. 5026-5036 ◽  
Author(s):  
Jun Liu ◽  
Tao Lin ◽  
Douglas J. Botkin ◽  
Erin McCrum ◽  
Hanspeter Winkler ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Electron Tomography ◽  
Rotational Symmetry ◽  
Flagellar Motor ◽  
Cell Axis ◽  
Ring Assembly ◽  
Flagellar Motors ◽  
Cryo Electron Tomography ◽  
Flagellar Rotation

ABSTRACT The bacterial flagellar motor is a remarkable nanomachine that provides motility through flagellar rotation. Prior structural studies have revealed the stunning complexity of the purified rotor and C-ring assemblies from flagellar motors. In this study, we used high-throughput cryo-electron tomography and image analysis of intact Borrelia burgdorferi to produce a three-dimensional (3-D) model of the in situ flagellar motor without imposing rotational symmetry. Structural details of B. burgdorferi, including a layer of outer surface proteins, were clearly visible in the resulting 3-D reconstructions. By averaging the 3-D images of ∼1,280 flagellar motors, a ∼3.5-nm-resolution model of the stator and rotor structures was obtained. flgI transposon mutants lacked a torus-shaped structure attached to the flagellar rod, establishing the structural location of the spirochetal P ring. Treatment of intact organisms with the nonionic detergent NP-40 resulted in dissolution of the outermost portion of the motor structure and the C ring, providing insight into the in situ arrangement of the stator and rotor structures. Structural elements associated with the stator followed the curvature of the cytoplasmic membrane. The rotor and the C ring also exhibited angular flexion, resulting in a slight narrowing of both structures in the direction perpendicular to the cell axis. These results indicate an inherent flexibility in the rotor-stator interaction. The FliG switching and energizing component likely provides much of the flexibility needed to maintain the interaction between the curved stator and the relatively symmetrical rotor/C-ring assembly during flagellar rotation.

scholarly journals Mechanics of torque generation in the bacterial flagellar motor

2015 ◽  
Vol 112 (32) ◽  
pp. E4381-E4389 ◽  
Author(s):  
Kranthi K. Mandadapu ◽  
Jasmine A. Nirody ◽  
Richard M. Berry ◽  
George Oster
Keyword(s):  
Conformational Changes ◽  
Specific Model ◽  
Power Stroke ◽  
Flagellar Motor ◽  
Mechanical Explanation ◽  
Proposed Model ◽  
Torque Generation ◽  
Bacterial Flagellar Motor ◽  
Α Helix ◽  
Fundamental Forces

The bacterial flagellar motor (BFM) is responsible for driving bacterial locomotion and chemotaxis, fundamental processes in pathogenesis and biofilm formation. In the BFM, torque is generated at the interface between transmembrane proteins (stators) and a rotor. It is well established that the passage of ions down a transmembrane gradient through the stator complex provides the energy for torque generation. However, the physics involved in this energy conversion remain poorly understood. Here we propose a mechanically specific model for torque generation in the BFM. In particular, we identify roles for two fundamental forces involved in torque generation: electrostatic and steric. We propose that electrostatic forces serve to position the stator, whereas steric forces comprise the actual “power stroke.” Specifically, we propose that ion-induced conformational changes about a proline “hinge” residue in a stator α-helix are directly responsible for generating the power stroke. Our model predictions fit well with recent experiments on a single-stator motor. The proposed model provides a mechanical explanation for several fundamental properties of the flagellar motor, including torque–speed and speed–ion motive force relationships, backstepping, variation in step sizes, and the effects of key mutations in the stator.

scholarly journals Mapping of Ebolavirus Neutralization by Monoclonal Antibodies in the ZMapp Cocktail Using Cryo-Electron Tomography and Studies of Cellular Entry

2016 ◽  
Vol 90 (17) ◽  
pp. 7618-7627 ◽  
Author(s):  
Erin E. H. Tran ◽  
Elizabeth A. Nelson ◽  
Pranay Bonagiri ◽  
James A. Simmons ◽  
Charles J. Shoemaker ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Conformational Changes ◽  
Neutralizing Antibodies ◽  
Structural Information ◽  
Electron Tomography ◽  
West African ◽  
Functional Studies ◽  
Cryo Electron Tomography ◽  
Niemann Pick ◽  
Viruslike Particles

ABSTRACTZMapp, a cocktail of three monoclonal antibodies (MAbs; c2G4, c4G7, and c13C6) against the ebolavirus (EBOV) glycoprotein (GP), shows promise for combatting outbreaks of EBOV, as occurred in West Africa in 2014. Prior studies showed that Fabs from these MAbs bind a soluble EBOV GP ectodomain and that MAbs c2G4 and c4G7, but not c13C6, neutralize infections in cell cultures. Using cryo-electron tomography, we extended these findings by characterizing the structures of c2G4, c4G7, and c13C6 IgGs bound to native, full-length GP from the West African 2014 isolate embedded in filamentous viruslike particles (VLPs). As with the isolated ectodomain, c13C6 bound to the glycan cap, whereas c2G4 and c4G7 bound to the base region of membrane-bound GP. The tomographic data suggest that all three MAbs bind with high occupancy and that the base-binding antibodies can potentially bridge neighboring GP spikes. Functional studies indicated that c2G4 and c4G7, but not c13C6, competitively inhibit entry of VLPs bearing EBOV GP into the host cell cytoplasm, without blocking trafficking of VLPs to NPC1+endolysosomes, where EBOV fuses. Moreover, c2G4 and c4G7 bind to and can block entry mediated by the primed (19-kDa) form of GP without impeding binding of the C-loop of NPC1, the endolysosomal receptor for EBOV. The most likely mode of action of c2G4 and c4G7 is therefore by inhibiting conformational changes in primed, NPC1-bound GP that initiate fusion between the viral and target membranes, similar to the action of certain broadly neutralizing antibodies against influenza hemagglutinin and HIV Env.IMPORTANCEThe recent West African outbreak of ebolavirus caused the deaths of more than 11,000 individuals. Hence, there is an urgent need to be prepared with vaccines and therapeutics for similar future disasters. ZMapp, a cocktail of three MAbs directed against the ebolavirus glycoprotein, is a promising anti-ebolavirus therapeutic. Using cryo-electron tomography, we provide structural information on how each of the MAbs in this cocktail binds to the ebolavirus glycoprotein as it is displayed—embedded in the membrane and present at high density—on filamentous viruslike particles that recapitulate the surface structure and entry functions of ebolavirus. Moreover, after confirming that two of the MAbs bind to the same region in the base of the glycoprotein, we show that they competitively block the entry function of the glycoprotein and that they can do so after the glycoprotein is proteolytically primed and bound to its intracellular receptor, Niemann-Pick C1. These findings should inform future developments of ebolavirus therapeutics.

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