Molecular mechanism for rotational switching of the bacterial flagellar motor

Abstract

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H⁺ driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.

Extended Data Fig. 1.. Swimming motility modes and flagellar switching in E. coli and B. burgdorferi.

(a, b) Cartoon of the swimming motility modes in E. coli: run and tumble. (c) The motor rotates CCW as a default state. (d) When the level of CheY-P becomes high enough, CheY-P binds to the C-ring, and the motor switches to CW rotation. The chemotaxis protein CheZ dephosphorylates CheY-P to return the motor to CCW rotation. (e, f) Swimming motility modes in B. burgdorferi: run and flex. Periplasmic flagella (PF) are located between the inner membrane (IM) and outer membrane (OM). The flagellar motors are attached near each cell pole. Spirochetes run when the anterior flagella rotate CCW and the posterior flagella rotate CW (e). When the flagella at both poles rotate in the same direction (CW), the spirochetes flex in place and fail to move translationally. The swimming motility of B. burgdorferi is also controlled by a chemotaxis system. The homologs of CheY and CheZ in B. burgdorferi are CheY3 and CheX.

Extended Data Fig. 2.. Cryo-ET imaging of the flagellar motors in ΔcheX and ΔcheY3 mutants.

(a) A representative tomographic section from a ΔcheX cell tip reconstruction. Outer membrane (OM), inner membrane (IM), peptidoglycan layer (PG), and motors are clearly resolved in the tomogram. (b) A representative section of a tomogram from a ΔcheY3 cell tip. Multiple motors with different orientations can be found at the cell tip. The insertions in (a, b) are the dark-field images showing a ΔcheX cell constantly flexing and a constantly running ΔcheY3 cell, respectively. (c) A medial cross-section of an averaged map of the ΔcheX motor. (d) The unrolled map refined using the stator region densities shows 16 stator complexes are embedded in the inner membrane (IM), while the C-ring subunits are unresolved due to symmetry mismatch between the C-ring and the stator. (e) The unrolled map refined using the C-ring region densities shows 46-fold symmetric features, while the stator becomes blurry. Bar = 20 nm.

Extended Data Fig. 3.. Comparison between in situ stator complex and the purified stator components.

(a) Structure of purified MotA complex from A. aeolicus resolved by single particle EM (EMD 3417). (b) The in situ stator complex in the ΔcheX motor has a bell-shaped structure embedded in the inner membrane (IM) and a periplasmic domain. The top part of the periplasmic domain matches well with the crystal structure of the S. enterica MotB periplasmic domain (PDB 2ZVY) (middle panel). The bell-shaped structure has similar size and shape as the structure of EMD 3417 (right panel). (c) The in situ stator complex in the ΔcheY3-Class-2 motor is similar to that in the ΔcheX motor.

Extended Data Fig. 4.. Schematic diagram for the in-frame replacement of cheX-cheY3 genes with cheY3-gfp.

(a) aadA, a streptomycin resistance gene was used as a selection marker. pcheX(F) and GFP (R) are oligonucleotide primers utilized to verify the occurrence of the allelic exchange of the recombinant construct (bottom) into the targeted region in the B. burgdorferi chromosome (top). (b) Ni-NTA affinity purification using FLAG-tagged FliM (FliM-FLAG) and FLAG-tagged FliN (FliN-FLAG) to pull down HisCheY3 or HisCheY3* (CheY3^D79A), respectively. HisCheY3 was co-purified with FliM-FLAG (b), but not with FliN-FLAG (c), suggesting that CheY3 does not bind on FliN. In contrast, more HisCheY3 protein was co-purified with FliM-FLAG with acetyl phosphate (b), and HisCheY3* was not co-purified with FliM-FLAG (a) or FliN-FLAG (c). These results indicate that CheY3 binds to FliM protein in a phosphorylation-dependent manner.

Extended Data Fig. 5.. Motor structures in constantly running ΔcheY3 cells.

(a) A medial cross-section of an averaged structure in ΔcheY3 B. burgdorferi cell. (b, c) Cross-sections show the stator ring and the C-ring, respectively. (d) Focused structure of the C-ring (unrolled along the central rod). Two distinct classes in the ΔcheY3 cells are named as ΔcheY3-Class-1 (e-h) and ΔcheY3-Class-2 (i-l). Class-1 and Class-2 account ~45% and ~55% of all the ΔcheY3 motors we used for current work, respectively. The stator structures in Class-1 and Class-2 (f and j) are quite similar, while the C-ring subunits (compare h with l) are tilted in different directions.

Extended Data Fig. 6.. Motors adopt distinct conformations at the two cell poles in the same ΔcheY3 cell.

(a) A tomographic section from one cell tip showed in panel b. (b) An overview of one intact ΔcheY3 cell. (c) A tomographic section from another tip of the same cell in panel b. The motors at each cell tip were aligned separately, then focused refined to the C-ring. (d-f) The motors from one cell tip have CCW conformation. (g-t) The motors from another tip appear to adopt CW conformation. (j-l) Averaged structure from motors located at one tip of five cells shows a better structure with CCW conformation. (m-o) Averaged structure from motors located at another tip of five cells shows a better structure with CW conformation. Bar = 200 nm in (a, c). Bar = 1 μm in (b).

Extended Data Fig. 7.. CheY3-P binding triggers conformational change.

(a-d) Comparison between the C-ring models before (grey, top left in each panel) and after (colored, top right in each panel) CheY3-P binding. (e) The dash framed regions in panel a are overlapped to show their differences. The N-terminal domain of FliM (FliM_N) folds out ~154° to interact with CheY3-P. (f) Binding of CheY3-P induces ~27° tilt of the FliM middle domain (FliM_M). (g, h) FliG2 undergoes a large tilt and alters the interactions between FliG2 and MotA. The charged residues (Lys275, Arg292, Glu299, and Asp300) in the C-terminal domain of FliG2 (FliG2_C) are colored in red.

Extended Data Fig. 8.. Comparison of the C-ring structures in CCW and CW rotation.

(a, b) Diameters of the FliG2_C, FliM and FliN rings in the C-ring with CCW rotation (ΔcheY3-class-2). (c-d) Diameters of the FliG2, FliM and FliN rings in the C-ring with CW rotation (ΔcheX).

Extended Data Fig. 9.. Motility model for B. burgdorferi.

(a, b) In the default state, the concentration of CheY3-P is low, and the cell runs. The motors at the anterior cell pole rotate CCW, and the motors at the posterior cell pole rotate CW. Binding of unidentified proteins (grey circles at the inner side of the C-ring) to the C-ring at the posterior cell pole likely changes the motor to a CW conformation. (c, d) At high concentrations of CheY3-P, the CCW rotating motors switch to CW rotation, while the CW rotating motors keep turning CW. Thus, the motors at both cell poles rotate CW and the cell flexes. After the flex, the direction of flagellar rotation at the two poles can switch so that the cell reverses the direction of its run.

Extended Data Fig. 10.. Oligonucleotide primers used in this study.

*F: forward; R, reverse. Underlined sequences are engineered restriction cut sites.

Figure 1.. Structure of the flagellar motor in constantly flexing ΔcheX cells.

(a) A medial cross-section of the in situ flagellar motor structure in ΔcheX determined by subtomogram averaging. The collar, stator, C-ring and export apparatus (EXP) are clearly visible in the cryo-ET map. (b) A perpendicular cross-section of the flagellar motor structure showing the stator ring. (c) The C-ring structure after focused alignment showing 46-fold symmetric features. (d-g) Stator-rotor interaction region (dash framed in panel a) after focused alignment. The arrowheads in (d) pointed out the unidentified densities associated with the C-ring. (e, g) The structures shown in (d and f) superimposed with the corresponding models in two different views. The unidentified densities associated with the C-ring were highlighted with grey and red circles in (e). (h) A top view of the stator ring on the top of the C-ring. (i) A side view of the flagellar motor structure in 3D. Scale bars, 20 nm.

Figure 2.. CheY3-P binding to the flagellar motor.

(a) Fluorescence image of cheX::cheY3-GFP cells showing that GFP-tagged CheY3 proteins are polarly localized. (b) A medial cross-section of the flagellar motor structure in cheX::cheY3-GFP cells. (c) A refined structure of the stator-rotor interface (dash framed in panel b) in cheX::cheY3-GFP. Extra density (green arrow) is associated with the C-ring. (d) A cartoon model is superimposed onto the structure shown in panel c. The GFP density (green arrow indicated in panel c and green color highlighted in d) is located outside the C-ring. (e, f) Ni-NTA affinity purifications using the poly-histidine modified proteins HisCheY3 or HisCheY3* (CheY3^D79A) to pull down FLAG-tagged FliM (FliM-FLAG) and FLAG-tagged FliN (FliN-FLAG), respectively. FliM-FLAG was co-purified with HisCheY3, but not with HisCheY3*, and more FliM-FLAG protein was co-purified with HisCheY3 in the presence of acetyl phosphate (e). There was no FliN-FLAG co-purified with HisCheY3/CheY3* (f). These results indicate that CheY3 binds to FliM protein in a phosphorylation-dependent manner. Scale bars, 10 μm in panel a, 20 nm in panel b.

Figure 3.. Stator-rotor interactions in constantly running ΔcheY3 cells.

Two distinct conformations of the C-ring are observed in ΔcheY3 cells. (a-e) Detailed motor conformation in the ΔcheY3-Class-1 with the same views as shown in Fig.1d–g, i. (f-j) Detailed motor structures in the ΔcheY3-Class-2. The C-ring appears different in two class averages. In Class-1, the C-ring interacts with the outer part of the stator; while in Class-2, the C-ring interacts with the inner part of the stator. (e, j) 3D surface views of the ΔcheY3-Class-1 and ΔcheY3-Class-2 flagellar motors. Note that the C-ring has two distinct conformations, enabling two different interactions with the stator complexes. Scale bar, 10 nm.

Figure 4.. Molecular architectures of the flagellar motors without and with CheY3-P.

(a) A medial cross-section of the flagellar motor structure without CheY3-P. (b) A pseudoatomic model of the C-ring unit shown in panel a. FliM and FliN have a stoichiometry of 1:3, and the FliM_C together with three FliN units form a spiral at the base of the C-ring. (c) Interactions between the bell-shaped stator complex and the C-ring. The charged residues (Lys275, Arg292, Glu299, and Asp300 in red) in FliG2_C interact with inner part of the stator complex. (d) A different view of five C-ring units connected at the based on the C-ring. (e) A medial cross-section of the flagellar motor structure in the presence of CheY3-P. (f) A pseudoatomic model of the C-ring unit with CheY3-P binding on the FliM_N. (g) The charged residues (Lys275, Arg292, Glu299, and Asp300 in red) in FliG2_C interact with outer part of the stator complex. (h) A different view of four C-ring units are occupied by four CheY3-P proteins.

Figure 5.. Model for the mechanism of rotational switching.

(a, b) Interactions of the stator with FliG2 in the C-ring during CCW rotation. In the default state when there is no bound CheY3-P, the FliG2 proteins interact with the inner part of the stator complex (colored in yellow). With the influx of protons through the stator channel, the cytoplasmic subunits of each stator complex spins CW. Therefore, the C-ring (blue) is induced to spin CCW. (c) A zoomed-in view of the interaction between the C-ring and the stator complex. (d) A perpendicular view shows that four C-ring units are connected by FliM/FliN interactions. (e, f) CheY3-P induced conformational changes in the C-ring result in altered interactions between the stator and C-ring, thereby causing the switch to CW rotation. When CheY3-P binds to FliM on the exterior surface of the C-ring, its binding triggers the shift (g) and tilt (h) of FliG2 so that FliG2_C interacts with the outer part of the cytoplasmic domain of the stator complex (g). Because the cytoplasmic domain of the stator always spins CW, the C-ring is induced to spin CW (e). During the rotational switch, the spiral ring structure formed by FliM and FliN acts as a base to hold the C-ring structure together (d, h).