Dataset Information

Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

ABSTRACT: The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays. Co-folding with FliFC induces substantial conformational changes in FliGN and suggests that FliF and FliG have the same stoichiometry within the rotor. Modeling the FliFC:FliGN complex into cryo-electron microscopy rotor density updates the architecture of the middle and upper switch complex and shows how domain shuffling of a conserved interaction module anchors the cytoplasmic rotor to the membrane.

SUBMITTER: Lynch MJ

PROVIDER: S-EPMC5387689 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

Lynch Michael J MJ Levenson Robert R Kim Eun A EA Sircar Ria R Blair David F DF Dahlquist Frederick W FW Crane Brian R BR

Structure (London, England : 1993) 20170112 2

The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliFC) bound to the N terminal domain of the switch complex protein FliG (FliGN) reveals that FliGN folds around FliFC to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The int ...[more]

PMID: 28089452

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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.

Project description:The bacterial flagellum is a complex, self-assembling macromolecular machine that powers bacterial motility. It plays diverse roles in bacterial virulence, including aiding in colonization and dissemination during infection. The flagellum consists of a filamentous structure protruding from the cell, and of the basal body, a large assembly that spans the cell envelope. The basal body is comprised of over 20 different proteins forming several concentric ring structures, termed the M- S- L- P- and C-rings, respectively. In particular, the MS rings are formed by a single protein FliF, which consists of two trans-membrane helices anchoring it to the inner membrane and surrounding a large periplasmic domain. Assembly of the MS ring, through oligomerization of FliF, is one of the first steps of basal body assembly. Previous computational analysis had shown that the periplasmic region of FliF consists of three structurally similar domains, termed Ring-Building Motif (RBM)1, RBM2, and RBM3. The structure of the MS-ring has been reported recently, and unexpectedly shown that these three domains adopt different symmetries, with RBM3 having a 34-mer stoichiometry, while RBM2 adopts two distinct positions in the complex, including a 23-mer ring. This observation raises some important question on the assembly of the MS ring, and the formation of this symmetry mismatch within a single protein. In this study, we analyze the oligomerization of the individual RBM domains in isolation, in the Salmonella enterica serovar Typhimurium FliF ortholog. We demonstrate that the periplasmic domain of FliF assembles into the MS ring, in the absence of the trans-membrane helices. We also report that the RBM2 and RBM3 domains oligomerize into ring structures, but not RBM1. Intriguingly, we observe that a construct encompassing RBM1 and RBM2 is monomeric, suggesting that RBM1 interacts with RBM2, and inhibits its oligomerization. However, this inhibition is lifted by the addition of RBM3. Collectively, this data suggest a mechanism for the controlled assembly of the MS ring.

Dataset Information

Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

Publications

Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

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