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

ABSTRACT: The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we show that the native MS ring is formed by 34 FliF subunits with no symmetry variation. Symmetry analysis of the C ring shows a variation with a peak at 34-fold, suggesting flexibility in C ring assembly. Finally, our data also indicate that FliF subunits assume two different conformations, contributing differentially to the inner and middle parts of the M ring and thus resulting in 23- and 11-fold subsymmetries in the inner and middle M ring, respectively. The internal core of the M ring, formed by 23 subunits, forms a hole of the right size to accommodate the protein export gate.

SUBMITTER: Kawamoto A

PROVIDER: S-EPMC8270960 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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

Kawamoto Akihiro A Miyata Tomoko T Makino Fumiaki F Kinoshita Miki M Minamino Tohru T Imada Katsumi K Kato Takayuki T Namba Keiichi K

Nature communications 20210709 1

The bacterial flagellar MS ring is a transmembrane complex acting as the core of the flagellar motor and template for flagellar assembly. The C ring attached to the MS ring is involved in torque generation and rotation switch, and a large symmetry mismatch between these two rings has been a long puzzle, especially with respect to their role in motor function. Here, using cryoEM structural analysis of the flagellar basal body and the MS ring formed by full-length FliF from Salmonella enterica, we ...[more]

PMID: 34244518

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Project description: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.

Project description: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 Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from 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 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 flagellar MS ring is formed by 34 subunits with 23-fold and 11-fold subsymmetries.

Publications

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

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