3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility
2022; Elsevier BV; Volume: 298; Issue: 7 Linguagem: Inglês
10.1016/j.jbc.2022.102105
ISSN1083-351X
AutoresSonia Mondino, Fabiana San Martin, Alejandro Buschiazzo,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoBacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way. Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way. Flagella are large supramolecular assemblies that work as nanomachines in bacteria, enabling the cells to swim in liquid environments at high speeds. In this way, bacteria move at ∼30 to 150 μm/s (1Turner L. Ryu William S. Berg Howard C. 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A 1 μm sized bacterium swimming at 100 μm/s (∼36 cm/h) advances ∼100 times its own body length per second, which is roughly equivalent to a 1.7 m sized person swimming at >600 km/h. As a reference, normal swimming speeds for an average human being are in the range of 3 km/h, and the fastest professional swimmers can attain ∼10 km/h. Bacterial flagella use energy very efficiently to turn on their motor and make it rotate at rates on the order of ∼100 to 270 Hz (6000–16,000 revolutions per minute) (5Berg H.C. The rotary motor of bacterial flagella.Annu. Rev. Biochem. 2003; 72: 19-54Crossref PubMed Scopus (1030) Google Scholar, 6Lowe G. Meister M. Berg H.C. Rapid rotation of flagellar bundles in swimming bacteria.Nature. 1987; 325: 637-640Crossref Google Scholar) or even up to sixfold faster in extreme cases (7Magariyama Y. Sugiyama S. Muramoto K. Maekawa Y. Kawagishi I. Imae Y. et al.Very fast flagellar rotation.Nature. 1994; 371: 752Crossref PubMed Google Scholar). Beyond motility, flagella mediate several processes, some of them relevant in host–pathogen interactions, including (i) biofilm formation and bacterial adherence to substrates, like host cells and tissues (8Haiko J. Westerlund-Wikstrom B. The role of the bacterial flagellum in adhesion and virulence.Biology. 2013; 2: 1242-1267Crossref PubMed Scopus (301) Google Scholar, 9Conrad J.C. Physics of bacterial near-surface motility using flagella and type IV pili: implications for biofilm formation.Res. Microbiol. 2012; 163: 619-629Crossref PubMed Scopus (68) Google Scholar); (ii) the secretion of virulence-associated proteins via the flagellar export apparatus (10Young G.M. Schmiel D.H. Miller V.L. A new pathway for the secretion of virulence factors by bacteria: the flagellar export apparatus functions as a protein-secretion system.Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6456-6461Crossref PubMed Scopus (0) Google Scholar); and (iii) immunomodulation and evasion from host immune response (11Chaban B. Hughes H.V. Beeby M. The flagellum in bacterial pathogens: for motility and a whole lot more.Semin. Cell Dev. Biol. 2015; 46: 91-103Crossref PubMed Scopus (167) Google Scholar). The structure and biogenesis of bacterial flagella, and the diverse array of biological roles they are involved in, have been the subject of excellent reviews (12Nedeljkovic M. Sastre D.E. Sundberg E.J. Bacterial flagellar filament: a supramolecular multifunctional nanostructure.Int. J. Mol. Sci. 2021; 22: 7521Crossref PubMed Scopus (7) Google Scholar, 13Erhardt M. Namba K. Hughes K.T. Bacterial nanomachines: the flagellum and type III injectisome.Cold Spring Harb. Perspect. Biol. 2010; 2a000299Crossref PubMed Scopus (167) Google Scholar, 14Nakamura S. Minamino T. Flagella-driven motility of bacteria.Biomolecules. 2019; 9: 279Crossref Scopus (109) Google Scholar), hence not further discussed in this article. Instead, this review is focused on a few selected motility functions that flagella are known to be engaged in, which have received recent attention because of the radical new mechanistic insights that have been uncovered. Double-membrane Gram-negative Enterobacteria, such as Salmonella enterica and Escherichia coli, have served over time as preferred models to study the structure and function of bacterial flagella, largely contributing to current bacterial motility paradigms (15Wadhwa N. Berg H.C. Bacterial motility: machinery and mechanisms.Nat. Rev. Microbiol. 2022; 20: 161-173Crossref PubMed Scopus (14) Google Scholar). The bacterial flagellum is organized into three basic substructures: (i) the basal body, which anchors the flagellum into the cell membrane and comprises a protein export apparatus, a rotary motor connected to a central drive shaft or rod, and bearing structures; (ii) the hook, which is directly joined to the rod and acts as a rotating universal joint coupling the motor to the filament along disparate axes; and (iii) the filament, an extremely long appendage that operates as a propeller generating thrust (16Evans L.D. Hughes C. Fraser G.M. Building a flagellum outside the bacterial cell.Trends Microbiol. 2014; 22: 566-572Abstract Full Text Full Text PDF PubMed Google Scholar) (Fig. 1). The entire flagellum is built with >25 different types of proteins, some of them present as single components, yet others repeated in tens of thousands of copies. In E. coli, each flagellum weighs ∼20 MDa, without considering the filament, which is on its own a ∼1.6 GDa polymer (comprising ∼30,000 flagellin protomers). In Gram-positive bacteria, most of the flagellar appendage is extracellular, with only part of the basal body including cytoplasmic and transmembrane structures (17Mukherjee S. Kearns D.B. The structure and regulation of flagella in Bacillus subtilis.Annu. Rev. Genet. 2014; 48: 319-340Crossref PubMed Scopus (86) Google Scholar). A similar cellular topology is exhibited by most Gram-negative species, with their basal body extending through the periplasm, the tip of their rod typically traversing the outer membrane, and their hook and filament exposed toward the extracellular milieu (18Altegoer F. Schuhmacher J. Pausch P. Bange G. From molecular evolution to biobricks and synthetic modules: a lesson by the bacterial flagellum.Biotechnol. Genet. Eng. Rev. 2014; 30: 49-64Crossref PubMed Scopus (21) Google Scholar). The flagellar apparatus is a fascinating illustration of evolutionary variation, sometimes leading to completely new functions based on conserved shared structures. A most extreme example of such a functional drift is the evolution of type-three secretion systems (T3SSs or injectisomes) from the flagellar ancestor (19Abby S.S. Rocha E.P. The non-flagellar type III secretion system evolved from the bacterial flagellum and diversified into host-cell adapted systems.PLoS Genet. 2012; 8e1002983Crossref PubMed Scopus (172) Google Scholar). The T3SS is a multiprotein assembly that has conserved many components of the flagellar protein export apparatus. Ultimately specializing in protein secretion, T3SSs lost several protein components, and with them, the abilities to rotate and drive motility altogether, while also evolving several unique elements to attain its exquisite protein secretion/injection capacities. We shall not address T3SS function and structural variations with further detail here, instead pointing the reader to several reviews on the subject (13Erhardt M. Namba K. Hughes K.T. Bacterial nanomachines: the flagellum and type III injectisome.Cold Spring Harb. Perspect. Biol. 2010; 2a000299Crossref PubMed Scopus (167) Google Scholar, 20Deng W. Marshall N.C. Rowland J.L. McCoy J.M. Worrall L.J. Santos A.S. et al.Assembly, structure, function and regulation of type III secretion systems.Nat. Rev. Microbiol. 2017; 15: 323-337Crossref PubMed Scopus (283) Google Scholar, 21Halte M. Erhardt M. Protein export via the type III secretion system of the bacterial flagellum.Biomolecules. 2021; 11: 186Crossref PubMed Scopus (1) Google Scholar, 22Diepold A. Armitage J.P. Type III secretion systems: the bacterial flagellum and the injectisome.Philos. Trans. R. Soc. B. 2015; 370: 20150020Crossref PubMed Scopus (0) Google Scholar). Less drastic architectural variations of rotating flagella are also observed, which can sometimes radically modify the locomotion mechanism. The hooks and filaments of several Gram-negative bacteria, such as Vibrio, Helicobacter, Brucella, and related genera, protrude extracellularly, yet are fully surrounded by the uninterrupted sheath of the outer membrane (23Chu J. Liu J. Hoover T.R. Phylogenetic distribution, ultrastructure, and function of bacterial flagellar sheaths.Biomolecules. 2020; 10: 363Crossref PubMed Scopus (6) Google Scholar). A more extreme modification is observed in the entire Spirochete phylum, where flagella, known in this group as endoflagella, are entirely confined within the periplasm, with the filament helically wrapped around the cell body (23Chu J. Liu J. Hoover T.R. Phylogenetic distribution, ultrastructure, and function of bacterial flagellar sheaths.Biomolecules. 2020; 10: 363Crossref PubMed Scopus (6) Google Scholar). Although such endoflagella are very similar to typical exoflagella regarding their architecture and protein composition, the fact that they evolved as an entirely periplasmic machine is driving an ongoing shift of the accepted bacterial motility paradigm. Even though further research is needed to fully elucidate the endoflagellar motility mechanism, it seems clear that their role involves a form of "dragging" the bacterial cell body and influencing cell morphology (24Wolgemuth C.W. Flagellar motility of the pathogenic spirochetes.Semin. Cell Dev. Biol. 2015; 46: 104-112Crossref PubMed Scopus (32) Google Scholar, 25Abe K. Kuribayashi T. Takabe K. Nakamura S. Implications of back-and-forth motion and powerful propulsion for spirochetal invasion.Sci. Rep. 2020; 10: 13937Crossref PubMed Scopus (3) Google Scholar). Accordingly, Spirochetes possess flagellar motors that can exert the highest torques so far observed in bacteria (26Beeby M. Ribardo D.A. Brennan C.A. Ruby E.G. Jensen G.J. Hendrixson D.R. Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E1917-E1926Crossref PubMed Scopus (108) Google Scholar), with the rest of the assembly particularly adapted to endure extreme rotation regimes within such a restricted volume of the cell. Torque is a vectorial quantity that represents the capability of a force to produce change in the rotational motion of a body. Analogous to a linear force's push and pull, torque can be thought of as a twist around a specific axis. It is the cross product of two vectors: (i) the distance of a rotating body—or a defined point in that rigid body—to the axis of rotation and (ii) the force applied to that body for it to rotate. Hence, the magnitudes of torque depend not only on the force applied but also on the length (radius) of the rotating object and the angle between them. These concepts will be important in appreciating the different means by which evolution has modulated higher or lower torques in different bacterial species. The ongoing revolutionary progress of three-dimensional cryo-EM and cryo–electron tomography (cryo-ET) approaches (27Bauerlein F.J.B. Baumeister W. Towards visual proteomics at high resolution.J. Mol. Biol. 2021; 433: 167187Crossref PubMed Scopus (0) Google Scholar, 28Nakane T. Kotecha A. Sente A. McMullan G. Masiulis S. Brown P. et al.Single-particle cryo-EM at atomic resolution.Nature. 2020; 587: 152-156Crossref PubMed Scopus (245) Google Scholar) has recently contributed to breakthrough observations of flagellar structures and their connection to the molecular mechanisms that underlie bacterial locomotion. In this review, we discuss major advances in structure–function relationships concerning the flagellar motor, hook, and filament, and future research directions that might answer some of the remaining open questions. We also highlight unique variations in Spirochete flagella, as a model to showcase how evolution uses an ancestral common structure to attain distinct mechanisms of translational motility in bacteria. The flagellar basal body harbors the motor device that drives flagellar rotation, while also including additional protein components that play key roles (Fig. 1): (i) a multiprotein export apparatus traversing the cell membrane and intruding into the cytoplasm, which ensures secretion of axial proteins during flagellar biogenesis; (ii) a set of stacked protein rings (C and MS rings), which constitute the moving rotor of the motor, and its switch complex that allows the direction of rotation to be inverted; (iii) several proteinaceous ring bearings (the P and L rings), isolating moving parts from fixed wall elements; and (iv) a hollow central shaft or rod, which connects the rotor to the hook. The rotor associates dynamically to the membrane-embedded and peptidoglycan (PG)-bound MotA–MotB stator units, which constitute the motor-force generators (29Zhao X. Norris S.J. Liu J. Molecular architecture of the bacterial flagellar motor in cells.Biochemistry. 2014; 53: 4323-4333Crossref PubMed Scopus (0) Google Scholar) (Fig. 1). In a general sense, motors are engines that perform mechanical work by using a two-component arrangement: a static part (stator) is fixed to a reference framework, whereas a moving part (rotor) rotates with respect to the stator. Some form of chemical and/or electrical input energy is needed to drive rotation unidirectionally. Brownian (random) back and forth small rotations because of thermal energy in the system would not produce effective work, as for the purpose of cell movement. For any form of energy to be useful at the nanometric scale of protein parts, such energy input must produce allosteric changes in transmission proteins, otherwise fuel consumption (which is too fast and localized) would be largely dissipated in the form of heat. For example, if ATP is hydrolyzed at a particular nucleotide-binding pocket within a motor, energy liberation from covalent bond breaking through phosphoryltransfer to water would dissipate as heat in a matter of picoseconds (30Henry E.R. Eaton W.A. Hochstrasser R.M. Molecular dynamics simulations of cooling in laser-excited heme proteins.Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8982-8986Crossref PubMed Google Scholar) and within a radius of a few Ångstroms (31Hwang W. Karplus M. Structural basis for power stroke vs. Brownian ratchet mechanisms of motor proteins.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 19777-19785Crossref PubMed Scopus (44) Google Scholar). These are meaningless ranges considering the protein motions that take place in biological nanomachines, which are measured in milliseconds and tens to hundreds of Ångstroms (32Yang W. Gao Y.Q. Cui Q. Ma J. Karplus M. The missing link between thermodynamics and structure in F1-ATPase.Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 874-879Crossref PubMed Scopus (94) Google Scholar, 33Karplus M. Gao Y.Q. Biomolecular motors: the F1-ATPase paradigm.Curr. Opin. Struct. Biol. 2004; 14: 250-259Crossref PubMed Scopus (0) Google Scholar). It is thus the protein rearrangements, linked to binding/dissociating ATP/ADP, that efficiently transduce the energy potential within the adequate time and space scales. In the bacterial flagellar motor, ATP hydrolysis does not drive rotation. Instead, it is the regulated transport of protons or sodium cations along their electrochemical gradient, from the outside to the inside of the cell. Such proton-motive potential is transduced to mechanical work via allosteric conformational/dynamic rearrangements of key motor proteins (34Hu H. Santiveri M. Wadhwa N. Berg H.C. Erhardt M. Taylor N.M.I. Structural basis of torque generation in the bi-directional bacterial flagellar motor.Trends Biochem. Sci. 2021; 47: 160-172Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). Recent cryo-EM data, obtained independently by two laboratories (35Deme J.C. Johnson S. Vickery O. Aron A. Monkhouse H. Griffiths T. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (56) Google Scholar, 36Santiveri M. Roa-Eguiara A. Kuhne C. Wadhwa N. Hu H. Berg H.C. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257.e16Abstract Full Text Full Text PDF PubMed Google Scholar), uncovered the molecular mechanism of such allosteric transitions, explaining with unprecedented detail how the energy is transduced through the stator to the rotor, forcing the flagellar motor to rotate. High-resolution 3D reconstructions of the stator piece of bacterial flagella were obtained by single-particle analysis (SPA). The stator complex comprises several units of two protein components, MotA and MotB (Fig. 1). For many years, the stator was thought to respect a 4:2 MotA:MotB stoichiometry (37Kojima S. Blair D.F. Solubilization and purification of the MotA/MotB complex of Escherichia coli.Biochemistry. 2004; 43: 26-34Crossref PubMed Google Scholar, 38Leake M.C. Chandler J.H. Wadhams G.H. Bai F. Berry R.M. Armitage J.P. Stoichiometry and turnover in single, functioning membrane protein complexes.Nature. 2006; 443: 355-358Crossref PubMed Scopus (433) Google Scholar). However, those data were deduced from well-designed 35S-radiolabeling biochemical experiments, which however did not visualize the assembled protein complex directly. Now, cryo-EM clearly reveals an "asymmetric" 5:2 MotA:MotB ratio, a slight yet extremely relevant difference to previous interpretations (37Kojima S. Blair D.F. Solubilization and purification of the MotA/MotB complex of Escherichia coli.Biochemistry. 2004; 43: 26-34Crossref PubMed Google Scholar). Furthermore, this 5:2 stoichiometry is conserved among many bacterial species, both Gram-negative (Vibrio mimicus and Campylobacter jejuni) and Gram-positive (Clostridium sporogenes and Bacillus subtilis), as well as across the entire family of MotA/MotB orthologs, such as PomA/PomB, the Ton transport ExbB/ExbD systems, and even the nonhomologous GldL/GldM gliding machines (35Deme J.C. Johnson S. Vickery O. Aron A. Monkhouse H. Griffiths T. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (56) Google Scholar). The asymmetry within the stator assembly appears to be intimately related to the very capacity of exerting unidirectional mechanical work. The two MotB monomers lie side by side, constituting an inner core, which is surrounded by a slightly distorted pentagon of MotA helices (Fig. 2A). The 5:2 ratio constrains each MotB monomer to sit into nonequivalent environments at any given time: each MotB helix is forced to interact with a different constellation of MotA residues. It is precisely this helix that includes the key cation transporter amino acid within MotB, namely aspartate 22 (according to the C. jejuni numbering scheme), a strictly conserved residue in bacterial MotB orthologs across phyla (Fig. 2A). The dissimilar environments play a central role in allowing for a "see-saw" alternating motion, of the carboxylate-bearing Asp22 side chains. The conformational change linked to the transport of each proton (a hydronium cation in water) is not very large, approximately 100-fold smaller than the estimated arc length traversed by the rotor (∼20–38 Å) per cation passage (36Santiveri M. Roa-Eguiara A. Kuhne C. Wadhwa N. Hu H. Berg H.C. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257.e16Abstract Full Text Full Text PDF PubMed Google Scholar). This feature is more consistent with a biased diffusion mechanism, also known as a Brownian ratchet, rather than one based on a power stroke. In the latter, the magnitude of the protein shifts is typically on the order of several nanometers, that is, distances comparable to the dimension of the protein components themselves (31Hwang W. Karplus M. Structural basis for power stroke vs. Brownian ratchet mechanisms of motor proteins.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 19777-19785Crossref PubMed Scopus (44) Google Scholar), with the swinging motion of myosin's lever arm being a prototypical example (39Sweeney H.L. Houdusse A. Structural and functional insights into the myosin motor mechanism.Annu. Rev. Biophys. 2010; 39: 539-557Crossref PubMed Scopus (289) Google Scholar). Directionally biased diffusion as a driver of mechanical movements (Fig. 2B), particularly as a means of powering a rotating motor device that produces effective work, received statistical thermodynamics support in the famous lecture "ratchet and pawl" by Richard Feynman (40Feynman R. Leighton R. Sands M. Ratchet and Pawl.in: The Feynman Lectures on Physics. Volume I. California Institute of Technology, New York, NY1963Google Scholar). Essentially, the asymmetric configuration of the ratchet and a certain input of energy to fix the pawl(s) impose a unidirectional sense to the otherwise random Brownian motion. A simple ratchet can be illustrated by a round gear with asymmetric teeth and one or more pawls as pivoting spring-loaded fingers engaging the teeth (Fig. 2B). The energy instilled onto the pawl counteracts the random motion, and the asymmetric interaction with the ratchet is maintained, obstructing reversed rotation (Fig. 2B). The recent cryo-EM data (35Deme J.C. Johnson S. Vickery O. Aron A. Monkhouse H. Griffiths T. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (56) Google Scholar, 36Santiveri M. Roa-Eguiara A. Kuhne C. Wadhwa N. Hu H. Berg H.C. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257.e16Abstract Full Text Full Text PDF PubMed Google Scholar) indeed demonstrate that the Brownian ratchet mechanism is at play within the stator complex itself. Two features deserve special attention (Fig. 2C): (i) the force injected to the aspartic acid/aspartates (the pawls) on MotB is due to the binding of protons on one side, and their dissociation on the other, along the electrochemical gradient; the Asp carboxylate–bearing side chains rearrange spatially, according to them being charged or neutral, alternating such shifts in a see-saw fashion between both MotB monomers; and (ii) the molecular surface of the MotA pentamer (the ratchet) is irregular and offers an asymmetric junction to the moving aspartic acid/aspartates. It is thus clear that MotA and MotB constitute a fully functional motor on their own. MotA acts as the moving rotor, whereas MotB is the stator, fixed to a static reference such as the PG through its C-terminal PG-binding OmpA domain. The interaction of the pentamer of MotA with the FliG multimer that constitutes the C ring allows the stator to transmit the rotary motion to the flagellar rotor (Fig. 1). In situ images of entire motors embedded within the inner membrane of Borrelia burgdorferi and Vibrio alginolyticus were recently obtained by cryo-ET at ∼20 Å resolution (41Carroll B.L. Nishikino T. Guo W. Zhu S. Kojima S. Homma M. et al.The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching.Elife. 2020; 9e61446Crossref Google Scholar, 42Chang Y. Zhang K. Carroll B.L. Zhao X. Charon N.W. Norris S.J. et al.Molecular mechanism for rotational switching of the bacterial flagellar motor.Nat. Struct. Mol. Biol. 2020; 27: 1041-1047Crossref PubMed Scopus (37) Google Scholar), demonstrating that MotA and FliG act as two coupled cogwheels. This mechanism allows to switch the sense of rotation of one of the cogwheels by inverting the direction of coupling between the two. While MotA always rotates clockwise (CW), FliG on the C ring can turn either CW or counter-clockwise (CCW). Flipping the FliG–MotA interface is enacted by a conformational rearrangement of the C-terminal domain of FliG, according to the states that the FliG-bound FliM–FliN switch complex adopts in response to stimuli (Fig. 1). Not only do the shape and molecular details of the purified stator complex (35Deme J.C. Johnson S. Vickery O. Aron A. Monkhouse H. Griffiths T. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (56) Google Scholar, 36Santiveri M. Roa-Eguiara A. Kuhne C. Wadhwa N. Hu H. Berg H.C. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257.e16Abstract Full Text Full Text PDF PubMed Google Scholar) fit consistently into the in situ cryo-ET volumes of the whole motor (41Carroll B.L. Nishikino T. Guo W. Zhu S. Kojima S. Homma M. et al.The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching.Elife. 2020; 9e61446Crossref Google Scholar, 42Chang Y. Zhang K. Carroll B.L. Zhao X. Charon N.W. Norris S.J. et al.Molecular mechanism for rotational switching of the bacterial flagellar motor.Nat. Struct. Mol. Biol. 2020; 27: 1041-1047Crossref PubMed Scopus (37) Google Scholar), but the latter images also lend strong support to the flipping mechanism and hence to the two-cogwheel gear model. The tomographic volumes were reconstructed from motors locked in the CW or CCW states, and the comparison of both uncovers a large conformational change of the C ring, whereas the stator positions remain unchanged. In the case of B. burgdorferi (42Chang Y. Zhang K. Carroll B.L. Zhao X. Charon N.W. Norris S.J. et al.Molecular mechanism for rotational switching of the bacterial flagellar motor.Nat. Struct. Mol. Biol. 2020; 27: 1041-1047Crossref PubMed Scopus (37) Google Scholar), the wall of the C ring closes its upper membrane-facing diameter relative to the motor axis to ∼55 nm in the CCW state, whereas opening it to ∼62 nm in the CW state. The same pattern of open/closure rearrangements also takes place in other species (41Carroll B.L. Nishikino T. Guo W. Zhu S. Kojima S. Homma M. et al.The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switchin
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