Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Kawamoto, Akihiro; Miyata, Tomoko; Makino, Fumiaki; Kinoshita, Miki; Minamino, Tetsuo; Imada, Katsumi; Kato, Takayuki; Namba, Keiichi

doi:10.1101/2020.10.11.334888

Cited by 10 publications

(33 citation statements)

References 52 publications

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“…The crystal structure of RBM1 and RBM2 together with the cryoEM structure of the MS-ring, including a high-resolution structure of the S-ring (RBM3), revealed the periplasmic part of the MS-ring structure. Our cryoEM analysis of the MS-FliG ring complex demonstrated that the MS-ring in the basal body is made up of 34 FliF subunits and is consistent with results of the recent cryoEM studies of the MS-ring formed by fulllength FliF and the MS-ring in the flagellar basal body (25). The S-ring with a cylindrical collar is shaped like a boater hat with C34 symmetry, whereas the inner part of the Mring has a gear wheel-like structure consisting of the core C23 symmetry inner ring and the C11 symmetry cogs surrounding it.…”

Section: Discussionsupporting

confidence: 90%

“…In addition, 21 or 22 copies of RBM2 form a ring in the inner part of the M-ring, and this is surrounded by 9 or 10 globular densities composed of RBM1 and RBM2. However, a few copies of RBM2 and more than 20 copies of RBM1 are missing in these structures, and the variation in the subunit stoichiometry is likely to be an artifact due to C-terminal truncations of FliF because the MS-ring in the flagellar basal body, as well as that formed by full-length recombinant FliF, showed only 34-fold rotational symmetry ( 25 ). Moreover, the cryoEM analysis of the purified the flagellar basal body showed a C23 symmetry at the inner part of the M-ring ( 25 ).…”

Section: Introductionmentioning

confidence: 99%

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Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring

Takekawa

Kawamoto

Sakuma

et al. 2021

mBio

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The bacterial flagellum is a protein nanomachine essential for bacterial motility. The flagellar basal body contains several ring structures. The MS-ring is embedded in the cytoplasmic membrane and is formed at the earliest stage of flagellar formation to serve as the base for flagellar assembly as well as a housing for the flagellar protein export gate complex. The MS-ring is formed by FliF, which has two transmembrane helices and a large periplasmic region. A recent electron cryomicroscopy (cryoEM) study of the MS-ring formed by overexpressed FliF revealed a symmetry mismatch between the S-ring and inner part of the M-ring. However, the actual symmetry relation in the native MS-ring and positions of missing domains remain obscure. Here, we show the structure of the M-ring by combining cryoEM and X-ray crystallography. The crystal structure of the N-terminal half of the periplasmic region of FliF showed that it consists of two domains (D1 and D2) resembling PrgK D1/PrgH D2 and PrgK D2/PrgH D3 of the injectisome. CryoEM analysis revealed that the inner part of the M-ring shows a gear wheel-like density with the inner ring of C23 symmetry surrounded by cogs with C11 symmetry, to which 34 copies of FliFD1–D2 fitted well. We propose that FliFD1–D2 adopts two distinct orientations in the M-ring relative to the rest of FliF, with 23 chains forming the wheel and 11 chains forming the cogs, and the 34 chains come together to form the S-ring with C34 symmetry for multiple functions of the MS-ring. IMPORTANCE The bacterial flagellum is a motility organelle formed by tens of thousands of protein molecules. At the earliest stage of flagellar assembly, a transmembrane protein, FliF, forms the MS-ring in the cytoplasmic membrane as the base for flagellar assembly. Here, we solved the crystal structure of a FliF fragment. Electron cryomicroscopy (cryoEM) structural analysis of the MS-ring showed that the M-ring and S-ring have different rotational symmetries. By docking the crystal structure of the FliF fragment into the cryoEM density map of the entire MS-ring, we built a model of the whole periplasmic region of FliF and proposed that FliF adopts two distinct conformations to generate three distinct C11, C23, and C34 symmetries within the MS-ring for its multiple functions.

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Section: Discussionsupporting

confidence: 90%

Section: Introductionmentioning

confidence: 99%

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring

Takekawa

Kawamoto

Sakuma

et al. 2021

mBio

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“…However, recent structural analysis of the MS ring revealed its symmetry to be 33-fold with a variation from 32 to 35 (Ref. 27 ), and the analysis of the flagellar basal body showed the MS ring to have 34-fold symmetry without variation and the C ring to have a small symmetry variation around 34-fold 28 . These results indicate the number of FliG on the C ring is mostly 34 and therefore invalidate the previous interpretation that the 26 steps per revolution reflect the elementary process of torque generation.…”

Section: Discussionmentioning

confidence: 99%

“…Although the LP ring is a stable component of the hook-basal body even after its isolation from the cell, the LP ring easily slips off the rod when the basal body is isolated without the hook 6,7,28 , indicating very weak interactions between the rod and LP ring to keep the LP ring in the proper position around the rod. In the absence of FlgH, however, the basal body formation stops just after completion of P ring assembly by FlgI around the rod to form a structure called “candlestick” 7,29 , indicating that the P ring is tightly attached to the rod until the L ring is assembled above it.…”

Section: Discussionmentioning

confidence: 99%

Structure of the molecular bushing of the bacterial flagellar motor

Yamaguchi

Makino

Miyata

et al. 2020

Preprint

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The bacterial flagellum is a motility organelle, consisting of the basal body acting as a rotary motor, the filament as a helical propeller and the hook connecting these two as a universal joint1,2. The basal body contains three rings: the MS ring as the transmembrane core of the rotor; the C ring essential for torque generation and switching regulation; and the LP ring as a bushing supporting the distal rod for its rapid, stable rotation without much friction. The negatively charged surface of the distal rod suggested electrostatic repulsive force in supporting high-speed rotation of the rod as a drive shaft3, but the LP ring structure was needed to see the actual mechanisms of its bushing function and assembly against the repulsive force. Here we report the LP ring structure by electron cryomicroscopy at 3.5 Å resolution, showing 26-fold rotational symmetry and intricate intersubunit interactions of each subunit with up to six partners that explains the structural stability. The inner surface is charged both positively and negatively, and positive charges on the P ring presumably play important roles in its initial assembly around the rod in the peptidoglycan layer followed by the L ring assembly in the outer membrane.

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“…The MS ring is constructed by assembling dozen copies of FliF, a protein with two transmembrane segments (10)(11)(12). The subatomic structure of the MS ring was determined by cryo-electron microscopy, although the N-terminal and C-terminal regions were not found in S. enterica (13,14). The C ring is composed of three proteins, FliG, FliM, and FliN, and forms a complex with the MS ring via FliG (15).…”

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confidence: 99%

Role of the N- and C-terminal regions of FliF, the MS ring component inVibrioflagellar basal body

Kojima

Kajino

Hirano

et al. 2021

Preprint

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The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

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Native structure of flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries

Cited by 10 publications

References 52 publications

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring

Two Distinct Conformations in 34 FliF Subunits Generate Three Different Symmetries within the Flagellar MS-Ring

Structure of the molecular bushing of the bacterial flagellar motor

Role of the N- and C-terminal regions of FliF, the MS ring component inVibrioflagellar basal body

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