Chemotaxis signaling protein CheY binds to the rotor protein FliN to control the direction of flagellar rotation in Escherichia coli - PubMed (original) (raw)

Chemotaxis signaling protein CheY binds to the rotor protein FliN to control the direction of flagellar rotation in Escherichia coli

Mayukh K Sarkar et al. Proc Natl Acad Sci U S A. 2010.

Abstract

The direction of rotation of the Escherichia coli flagellum is controlled by an assembly called the switch complex formed from multiple subunits of the proteins FliG, FliM, and FliN. Structurally, the switch complex corresponds to a drum-shaped feature at the bottom of the basal body, termed the C-ring. Stimulus-regulated reversals in flagellar motor rotation are the basis for directed movement such as chemotaxis. In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. In response to the chemotaxis signaling molecule phospho-CheY (CheY(P)), the motors can switch to clockwise (CW) rotation, causing dissociation of the filament bundle and reorientation of the cell. CheY(P) has previously been shown to bind to a conserved segment near the N terminus of FliM. Here, we show that this interaction serves to capture CheY(P) and that the switch to CW rotation involves the subsequent interaction of CheY(P) with FliN. FliN is located at the bottom of the C-ring, in close association with the C-terminal domain of FliM (FliM(C)), and the switch to CW rotation has been shown to involve relative movement of FliN and FliM(C). Using a recently developed structural model for the FliN/FliM(C) array, and the CheY(P)-binding site here identified on FliN, we propose a mechanism by which CheY(P) binding could induce the conformational switch to CW rotation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Electron microscopic images of the flagellar basal body, from studies in Salmonella (10). (A) Single-particle reconstruction of the full basal body, including the LP-ring and a short segment of the rod. The structure is viewed from the side and has been axially averaged. The MS-ring is at the level of the cytoplasmic membrane; the C-ring is in the cytosol. The diameter of the C-ring is about 50 nm. A current working hypothesis for the locations of FliG, FliM, and FliN (–17) is shown at the right of the C-ring. The two lobes of density at the top of the C-ring are both assigned to FliG, with the outer one corresponding to the C-terminal domain. [In a more fully detailed model, some of the FliM subunits are hypothesized to tilt inward to interact with the inner domain of FliG (13), but this feature is not important in the present context.] The inward-pointing extension on FliM represents the N-terminal segment that is known to interact with CheYP. (B) Detail from a higher-resolution reconstruction (11), showing rings of density at the bottom of the C-ring. [Reproduced with permission from Thomas D, DeRosier DJ (2001) (Copyright 2010, American Society for Microbiology).] (C) The appearance of the bottom of the C-ring as determined in the high-resolution reconstruction and the organization of FliN tetramers and FliMC domains at the bottom of the C-ring as deduced from cross-linking and mutational studies (15).

Fig. 2.

Fig. 2.

(A) Schematic of the FliM1–34–CheY fusion construct. The part of the FliM sequence shown explicitly is well conserved across species and is known to bind to CheYP in helical conformation (23). (B Left) Pull-down assay with GST–FliN and the FliM1–34–CheY construct, in presence or absence of the phosphorylating agent acetyl phosphate. (B Right) Effect of the CheY mutation D57A. (C) Binding of the FliM1–34–CheY construct to FliN proteins with mutations in various surface positions. Acetyl phosphate was present in all samples. (D) Comparison of the CheYP-binding region on FliN with positions of previously characterized CCW-biased mutations. (Left) Results of the binding experiment (C) mapped onto the FliN structure (PDB ID code 1yab). Red, positions where mutations eliminated the binding; blue, positions where mutations did not affect binding. (Right) Positions of mutations in FliN that gave CCW motor bias, colored green (data from ref. ; image made in PyMol).

Fig. 3.

Fig. 3.

Effect of mutations in the segment of FliM that interacts with CheY. (A) Structure of the complex formed between CheY (light gray) and the FliM segment (yellow) (PDB ID code 1f4v) (23). The solvent-exposed residues Ala-9 and Glu-10 are colored cyan, and residues Gln-8 and Asn-16, which contribute to the FliM–CheY interface, are magenta (image made in PyMol) (B) Interaction of FliN with FliM1–34–CheY constructs with mutations in the FliM segment. The pull-down assay used GST–FliN and the FliM1–34–CheY fusion construct containing the mutations. (C) Effects of the FliM mutations on cell migration in soft agar. The Trp replacements were transferred into the full-length FliM protein for this experiment. (D) Effects of the FliM mutations on the interaction between the FliM segment and CheY, measured using a GST–CheY pull-down assay.

Fig. 4.

Fig. 4.

Effect of cross-linking through the N-terminal segment of FliN on the binding of FliN to the FliM1–34–CheY construct. Residue Trp-19 of FliN (in the GST–FliN fusion) was replaced with Cys. In the samples indicated, Cu-Tris[1,10-phenanthroline] was added to induce disulfide formation.

Fig. 5.

Fig. 5.

Model of CheYP-induced flagellar motor switching. (A Left) CheYP (yellow) interacts initially with the N-terminal segment of FliM, which is flexible enough to allow subsequent binding to a site on FliN (orange) in the vicinity of the hydrophobic patch. Only the FliM and FliN proteins of the switch are shown; FliG would be at the top (Fig. 1). (Right) View showing multiple FliM–FliN units in the lower part of the C-ring, and the binding of multiple CheYP molecules. (B) Relationship of FliN4 and FliMC units in the bottom of the C-ring, as determined from cross-linking and mutational analysis (15). One FliN4–FliMC–FliN4 unit is shown, in stereoview. Altered yields of certain cross-links upon switching (15) indicated that motor reversal is accompanied by a movement along one of the FliN4–FliMC interfaces, shown here by the two locations for the left-hand FliN4 unit (CW state, gray; CCW state, cyan). The CheYP-binding site on FliN is colored orange. (C) Hypothesis for switching in all of the FliN4–FliMC units, shown in top view. CheYP molecules are yellow and binding sites are orange; the black dot signifies the N-terminal segment of FliM sandwiched between CheYP and FliN. These segments would attach to the FliM middle domain, by a flexible linker (A).

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