Structural basis of torque generation in the bi-directional bacterial flagellar motor
2021; Elsevier BV; Volume: 47; Issue: 2 Linguagem: Inglês
10.1016/j.tibs.2021.06.005
ISSN1362-4326
AutoresHaidai Hu, Mònica Santiveri, Navish Wadhwa, Howard C. Berg, Marc Erhardt, Nicholas M. I. Taylor,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoThe bacterial flagellum is a supramolecular machine essential for locomotion and virulence of many bacteria and comprises a long filament connected through a hook to a cell-enveloped embedded basal body.The basal body comprises a bi-directional rotary motor energized by the stator units that surround it; the stator unit harnesses the electrochemical gradient of ions across the cytoplasmic membrane to generate torque.Cryo-electron microscopic (cryo-EM) structures of the stator unit revealed its stoichiometry (a MotA pentamer surrounding a MotB dimer), its autoinhibition mechanism, the ion flux pathway, and the conformational changes upon protonation driving MotA rotation.Phosphorylated chemotaxis signaling protein CheY-P binds to the C-ring of the flagellar motor, inducing a conformational change that alters the interaction between C-ring and stator units, switching the rotational direction of the flagellar motor from counterclockwise to clockwise. The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor. The flagellar stator unit is an oligomeric complex of two membrane proteins (MotA5B2) that powers bi-directional rotation of the bacterial flagellum. Harnessing the ion motive force across the cytoplasmic membrane, the stator unit operates as a miniature rotary motor itself to provide torque for rotation of the flagellum. Recent cryo-electron microscopic (cryo-EM) structures of the stator unit provided novel insights into its assembly, function, and subunit stoichiometry, revealing the ion flux pathway and the torque generation mechanism. Furthermore, in situ cryo-electron tomography (cryo-ET) studies revealed unprecedented details of the interactions between stator unit and rotor. In this review, we summarize recent advances in our understanding of the structure and function of the flagellar stator unit, torque generation, and directional switching of the motor. Many bacteria, including Escherichia coli, Salmonella, and Bacillus spp., use flagella (see Glossary) to move through liquid environments and across surfaces. The flagellum is a supramolecular nanomachine that protrudes from the cell envelope and measures ~5–20 μm in length. It is able to rotate in both clockwise (CW) and counterclockwise (CCW) directions to propel the bacterial cell body in different living environments [1.Nakamura S. Minamino T. Flagella-driven motility of bacteria.Biomolecules. 2019; 9: 279Crossref Scopus (80) Google Scholar,2.Armitage J.P. Berry R.M. Assembly and dynamics of the bacterial flagellum.Annu. Rev. Microbiol. 2020; 74: 181-200Crossref PubMed Scopus (9) Google Scholar]. Rotational switching between these two modes is regulated by chemotactic signaling, which is a rapid process that responds to environmental stimuli and biases movement of the cell toward attractants and away from repellents. 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The MS-ring, which comprises multiple copies of the transmembrane protein FliF [17.Johnson S. et al.Molecular structure of the intact bacterial flagellar basal body.Nat. Microbiol. 2021; 6: 712-721Crossref PubMed Scopus (0) Google Scholar,27.Kawamoto A. et al.Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries.bioRxiv. 2020; (Published online October 11, 2020. https://doi.org/10.1101/2020.10.11.334888)Google Scholar], forms a structural scaffold around the flagellar export apparatus and coordinates the formation of the C-ring, which engages with the stator units to generate torque [28.Terashima H. et al.Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species.J. Bacteriol. 2020; (Published online June 1, 2020. https://doi.org/10.1128/JB.00236-20)Crossref Scopus (2) Google Scholar]. The stator unit is a complex of two membrane proteins sharing the same operon on the genomic locus, with a molecular mass of ~200 kDa [29.Liu R. Ochman H. Origins of flagellar gene operons and secondary flagellar systems.J. Bacteriol. 2007; 189: 7098-7104Crossref PubMed Scopus (47) Google Scholar]. Located at the inner membrane, the stator unit is responsible for harvesting the cross-membrane electrochemical gradient of ions, most commonly protons or sodium ions (e.g., MotA/MotB is a H+-dependent stator unit; PomA/PomB is a Na+-dependent stator unit), while, in some cases, the stator unit also uses potassium and rubidium ions [30.Terahara N. et al.A Bacillus flagellar motor that can use both Na+ and K+ as a coupling ion is converted by a single mutation to use only Na+.PLoS ONE. 2012; 7e46248Crossref PubMed Scopus (32) Google Scholar]. Some bacterial species contain only one type of stator unit, whereas others have multiple types [31.Paulick A. et al.Dual stator dynamics in the Shewanella oneidensis MR-1 flagellar motor.Mol. Microbiol. 2015; 96: 993-1001Crossref PubMed Scopus (41) Google Scholar]. For example, Vibrio alginolyticus contains only sodium-driven stator units and Campylobacter jejuni contains only proton-driven stator units, while Bacillus subtilis has both types [32.Minamino T. et al.Autonomous control mechanism of stator assembly in the bacterial flagellar motor in response to changes in the environment.Mol. Microbiol. 2018; 109: 723-734Crossref PubMed Scopus (20) Google Scholar]. In all stator units, one component is anchored to the bacterial cell wall, while the other component engages with the C-ring of the flagellar motor, thereby enabling torque generation. The stator unit is considered as a motor itself: it converts the electrochemical potential energy from the ion motive force into mechanical torque. Upon recruitment to the basal body and cell wall binding, the stator units undergo a conformational change from an inactive/plugged state into an activated/unplugged state [33.Hosking E.R. et al.The Escherichia coli MotAB proton channel unplugged.J. Mol. Biol. 2006; 364: 921-937Crossref PubMed Scopus (105) Google Scholar]. In the unplugged state, the flux of ions through the stator unit channel energizes the rotation of the rotor. Cryo-EM structures of stator units from different bacterial species have recently been determined to high resolution and their interactions with their rotors have been explored by cryo-tomographic studies [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar, 35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar, 36.Chang Y. et al.Structural insights into flagellar stator–rotor interactions.eLife. 2019; 8e48979Crossref PubMed Scopus (18) Google Scholar, 37.Carroll B.L. et al.The flagellar motor of Vibrio alginolyticus undergoes major structural remodeling during rotational switching.eLife. 2020; 9e61446Crossref PubMed Google Scholar, 38.Chang Y. et al.Molecular mechanism for rotational switching of the bacterial flagellar motor.Nat. Struct. Mol. Biol. 2020; 27: 1041-1047Crossref PubMed Scopus (18) Google Scholar]. Here, we focus on recent developments in the understanding of the flagellar stator unit, the biological mechanism of its torque generation, and the rotational switching of the motor. The C-terminal part of MotB or PomB [known as the peptidoglycan-binding (PGB) domain] allows binding of the stator unit to the peptidoglycan layer of the bacterial cell wall. The PGB domain displays a high degree of similarity to the C-terminal domain of OmpA, a flexible clamp responsible for bacterial cell wall binding [39.Samsudin F. et al.OmpA: a flexible clamp for bacterial cell wall attachment.Structure. 2016; 24: 2227-2235Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar]. High-resolution structures of isolated MotB PGB obtained through X-ray crystallography provide a wealth of information for a mechanistic understanding of its self-dimerization and its interaction with peptidoglycan components [40.Roujeinikova A. Crystal structure of the cell wall anchor domain of MotB, a stator component of the bacterial flagellar motor: Implications for peptidoglycan recognition.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 10348-10353Crossref PubMed Scopus (106) Google Scholar, 41.Zhu S. et al.Conformational change in the periplamic region of the flagellar stator coupled with the assembly around the rotor.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 13523-13528Crossref PubMed Scopus (0) Google Scholar, 42.O'Neill J. et al.Role of the MotB linker in the assembly and activation of the bacterial flagellar motor.Acta Crystallogr. D Biol. Crystallogr. 2011; 67: 1009-1016Crossref PubMed Scopus (0) Google Scholar]. The intrinsic dynamic properties of the stator unit probably precluded crystallization of the full complex. The first available 3D structure of a stator unit was that of PomAB from V. alginolyticus, reconstructed by single-particle analysis from negatively stained samples, with a resolution limited to ~20 Å [43.Yonekura K. et al.Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation.J. Bacteriol. 2011; 193: 3863-3870Crossref PubMed Scopus (36) Google Scholar]. The map revealed the overall shape of the stator unit, suggesting that two PomB molecules were surrounded by four PomA monomers, consistent with previous studies establishing the presence of at least two PomB and an apparent PomA:PomB ratio of ~2:1 [44.Sato K. Homma M. Functional reconstitution of the Na+-driven polar flagellar motor component of Vibrio alginolyticus.J. Biol. Chem. 2000; 275: 5718-5722Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar,45.Braun T.F. Blair D.F. Targeted disulfide cross-linking of the MotB protein of Escherichia coli: evidence for two H(+) channels in the stator complex.Biochemistry. 2001; 40: 13051-13059Crossref PubMed Scopus (99) Google Scholar]. This model was widely used as template for molecular dynamic simulations of ion transportation and amino acid point mutagenesis for functional studies [43.Yonekura K. et al.Structure of the flagellar motor protein complex PomAB: implications for the torque-generating conformation.J. Bacteriol. 2011; 193: 3863-3870Crossref PubMed Scopus (36) Google Scholar,46.Nishikino T. et al.Characterization of PomA periplasmic loop and sodium ion entering in stator complex of sodium-driven flagellar motor.J. Biochem. (Tokyo). 2020; 167: 389-398Crossref PubMed Google Scholar, 47.Nishihara Y. Kitao A. Gate-controlled proton diffusion and protonation-induced ratchet motion in the stator of the bacterial flagellar motor.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 7737-7742Crossref PubMed Google Scholar, 48.Takekawa N. et al.The tetrameric MotA complex as the core of the flagellar motor stator from hyperthermophilic bacterium.Sci. Rep. 2016; 6: 31526Crossref PubMed Scopus (21) Google Scholar]. However, due to the low resolution of the structure, it was not possible to accurately interpret the stator unit stoichiometry and channel formation. With the resolution revolution of single-particle cryo-EM, it became possible to determine high-resolution structures of membrane proteins without crystallization and with a smaller quantity of protein sample [49.Kühlbrandt W. The resolution revolution.Science. 2014; 343: 1443-1444Crossref PubMed Scopus (557) Google Scholar,50.Cheng Y. Membrane protein structural biology in the era of single particle cryo-EM.Curr. Opin. Struct. Biol. 2018; 52: 58-63Crossref PubMed Scopus (58) Google Scholar]. Both the relatively small molecular mass of the flagellar stator units as well as the preferred orientation that these particles adopt on EM grids have hindered their structural determination. By optimizing protein purification procedures and cryo-EM grid preparation, atomic models of the proton-driven MotAB stator unit family from three bacterial species were constructed [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. These studies have contributed detailed structural information about the subunit assembly and proposed a mechanism for stator unit activation and torque generation. The structures revealed that the stator unit adopts a MotA5:MotB2 arrangement. The 5:2 stoichiometry was also reinforced by low-resolution maps of two sodium-driven stator units, V. alginolyticus PomAB and Vibrio mimicus PomAB, albeit lacking the atomic coordinates [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. These data suggest a conserved arrangement across all types of flagellar stator unit: a pentamer of MotA peripherally surrounding a dimer of MotB (Figure 2A,C ). Of note, all three models lack the MotB C-terminal PGB domain, reflecting the highly flexible locations of MotB PGB with respect to the core structure, at least in a detergent environment [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. Moreover, the structures suggest that the stator unit is in an autoinhibited state (discussed later). Other evolutionarily related bacterial complexes, which also harness the transmembrane proton motive force, share the same 5:2 stoichiometry [51.Lai Y.-W. et al.Evolution of the stator elements of rotary prokaryote motors.J. Bacteriol. 2020; 202e00557-19Crossref PubMed Scopus (5) Google Scholar,52.Ratliff A.C. et al.Ton motor complexes.Curr. Opin. Struct. Biol. 2021; 67: 95-100Crossref PubMed Scopus (1) Google Scholar]. These include ExbB5D2, which powers the ExbB–ExbD–TonB complex, responsible for transportation of nutrients entering into the periplasmic space [53.Celia H. et al.Cryo-EM structure of the bacterial Ton motor subcomplex ExbB–ExbD provides information on structure and stoichiometry.Commun. Biol. 2019; 2: 1-6Crossref PubMed Scopus (16) Google Scholar], and GldL5M2, which powers the gliding motility/type 9 protein secretion system motors in members of the phylum Bacteroidetes [54.Hennell James R. et al.Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility.Nat. Microbiol. 2021; 6: 221-233Crossref PubMed Scopus (2) Google Scholar]. Among the different stator units studied, the structure of MotAB has been determined from C. jejuni (CjMotAB), a common foodborne pathogenic bacterium [55.Young K.T. et al.Campylobacter jejuni: molecular biology and pathogenesis.Nat. Rev. Microbiol. 2007; 5: 665-679Crossref PubMed Scopus (499) Google Scholar], to a resolution of 3.1 Å (with a local resolution as high as 2.5 Å) [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. Briefly, the transmembrane segments (TM) of MotA fold into α helices, with the third and fourth segments (TM3 and TM4) lining the dimerized MotB TM helices. MotA TM1 and TM2 establish extensive hydrophobic interactions with the lipid bilayer. Two amphipathic helices of MotA, the cytoplasmic interface helix (CI) and the periplasmic interface helix (PI), perpendicular to the TM3 and TM4, adopt a parallel orientation with reference to the membrane, clearly defining the membrane boundary of the stator unit (Figure 2A,B); this is consistent with the structures of the MotAB stator units from B. subtilis and Clostridium sporogenes [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. At the periplasmic interface, a short helix just after the TM of MotB, designated as a plug motif, wedges in between the top of two MotA subunits, revealing the autoinhibition mechanism of the stator unit [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar] (Figure 2B). The plug motifs of the two MotB chains are organized in a trans mode in the stator unit, consistent with earlier functional experiments [33.Hosking E.R. et al.The Escherichia coli MotAB proton channel unplugged.J. Mol. Biol. 2006; 364: 921-937Crossref PubMed Scopus (105) Google Scholar] (Figure 3A ). The density around the first ten residues of the MotB N terminus is less defined, preventing model building, suggesting that this region adopts different conformations [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. Additionally, the unplugged structure of CjMotAB (MotB ∆41–60) mimicking the active state of the stator unit, and the unplugged and protonated structure of CjMotAB (MotB ∆41–60, D22N) were characterized to a high resolution [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar] (Figure 3B). Given that the architecture and the sequence of the stator units are so well conserved, these high-resolution structures offer a great opportunity to understand how ions flow through the stator unit and induce rotation of MotA around MotB. One distinctive, universally conserved feature in MotB is the plug motif. Early functional experiments showed that overexpression of MotAB from E. coli did not impair cell growth [56.Stolz B. Berg H.C. Evidence for interactions between MotA and MotB, torque-generating elements of the flagellar motor of Escherichia coli.J. Bacteriol. 1991; 173: 7033-7037Crossref PubMed Scopus (120) Google Scholar]. By contrast, in-frame deletion of this plug motif leads to proton leakage and cell growth arrest, showing that activation of the MotAB channel is controlled by the plug motif [33.Hosking E.R. et al.The Escherichia coli MotAB proton channel unplugged.J. Mol. Biol. 2006; 364: 921-937Crossref PubMed Scopus (105) Google Scholar,57.Morimoto Y.V. et al.Proton-conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella.FEBS Lett. 2010; 584: 1268-1272Crossref PubMed Scopus (52) Google Scholar]. As noted earlier, the structure of the unplugged state of CjMotAB was obtained by deleting the residues corresponding to the plug motif [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. Interestingly, it was observed that the unplugged CjMotAB was toxic to E. coli cells when overexpressed [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. This unplugged structure unveils a potential proton channel that links the periplasmic space to the conserved acidic residue D22 on TM helix of MotB chain 2 (Figure 3D) (numbering to reflect the specific local environment of each segment in the asymmetric complex), and to the inside of MotA cytoplasmic domain, where many negatively charged residues are found [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. Inspection of the channel-lining residues reveals the differential conservation between proton and sodium-dependent stator units, which have previously been shown to be critical for ion transport (Figure 3C) [58.Onoue Y. et al.Essential ion binding residues for Na + flow in stator complex of the Vibrio flagellar motor.Sci. Rep. 2019; 9: 11216Crossref PubMed Scopus (0) Google Scholar, 59.Sudo Y. et al.Comparative study of the ion flux pathway in stator units of proton- and sodium-driven flagellar motors.Biophysics. 2009; 5: 45-52Crossref Scopus (16) Google Scholar, 60.Terauchi T. et al.A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque.Microbiology. 2011; 157: 2422-2432Crossref PubMed Scopus (20) Google Scholar]. In addition, this channel is shielded by the conserved hydrophobic residue F186 of TM4 of MotA, the side chain of which adopts two different conformations [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. Consequently, this residue is likely to be a key point controlling the ion flux, ensuring efficient ion motive force utilization. D22 on the TM of CjMotB chain 1 is buried in a hydrophobic environment; therefore, it is more likely to accommodate a protonated (or hydronium-interacting) D22 compared with the MotB chain 2 [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar]. Supporting this model, the proton channel is not observed in any of the three plugged stator units [34.Santiveri M. et al.Structure and function of stator units of the bacterial flagellar motor.Cell. 2020; 183: 244-257Abstract Full Text Full Text PDF PubMed Google Scholar,35.Deme J.C. et al.Structures of the stator complex that drives rotation of the bacterial flagellum.Nat. Microbiol. 2020; 5: 1553-1564Crossref PubMed Scopus (30) Google Scholar]. The comparison between plugged and unplugged MotAB structures reveals no major conformational differences, which argues against the idea that a large conformational change within MotA, without rotation of MotA around MotB, causes torque generation, which was the previ
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