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Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM

Abstract

Bacterial locomotion by rotating flagella is achieved through the hook, which transmits torque from the motor to the filament. The hook is a tubular structure composed of a single type of protein, yet it adopts a curved shape. To perform its function, it must be simultaneously flexible and torsionally rigid. The molecular mechanism by which chemically identical subunits form such a dynamic structure is unknown. Here, we show the complete structure of the hook from Salmonella enterica in its supercoiled ‘curved’ state, at 2.9 Å resolution. Subunits in the curved hook are grouped into 11 distinctive conformations, each shared along 11 protofilaments. The domains of the elongated hook subunit behave as rigid bodies connected by two hinge regions. The reconstituted model demonstrates how identical subunits can dynamically change conformation by physical interactions while bending. These multiple subunit states contradict the two-state model, which is a key feature of flagellar polymorphism.

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Fig. 1: Cryo-EM of polyhooks from Salmonella enterica.
Fig. 2: Domain motions of subunits around helical turns.
Fig. 3: Subunit interactions in the curved hook.

Data availability

The cryo-EM map of the curved hook has been deposited in the Electron Microscopy Data Bank under accession no. EMD-9909. The atomic coordinates of all 66 subunit models have been deposited in the wwPDB as a single file in mmCIF format under accession no. 6K3I. Source data for Fig. 2c are available with the online version of this paper.

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Acknowledgements

We thank M. Bandi for helpful discussions about the mechanical function of the hook and S.D. Aird for technical editing of the manuscript. This work was supported by the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED, under grant nos. 19am0101076 and 19am0101116 (to M.W.), by JSPS KAKENHI grants 17K17085 and 19K10083 (to S.S.) and by a JSPS KAKENHI grant 17K07318 (to H.M.). M.W. was supported by direct funding from the Okinawa Institute of Science and Technology Graduate University.

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Authors

Contributions

S.S., H.M., S.-I.A. and M.W. designed the experiments. S.S. and S.-I.A. purified the polyhooks. S.S. prepared cryo-EM specimens. S.S. and M.W. collected cryo-EM data and performed image processing. H.M. built and refined the atomic models. S.S. and H.M analyzed the structure and created figures. M.W. supervised the project. S.-I.A. and M.W. wrote the initial manuscript. All authors discussed the results and contributed to writing the paper.

Corresponding authors

Correspondence to Shin-Ichi Aizawa or Matthias Wolf.

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The authors declare no competing interests.

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Peer review information Ines Chen was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Integrated supplementary information

Supplementary Figure 1 Polyhooks in negative stain.

Electron micrograph of negatively stained (uranyl acetate) sonicated polyhooks on carbon support film. The protein can form flat ring-like structures under these conditions. Scale bar, 100 nm.

Supplementary Figure 2 Resolution estimation.

The reconstructed electron potential map clearly resolves side chain rotamers. The map was visualized with an iso-electron potential surface contoured at 2.6σ above average. a, N-terminal α-helix. The N terminus is at the bottom. b, C-terminal α-helix. The C terminus is at the bottom. Scale bar, 10 Å. c, A Fourier shell correlation calculated with the program cisTEM between independently refined maps, each containing half of the particle images, indicates a spatial resolution of 2.9 Å (at FSC = 0.143).

Supplementary Figure 3 Polyhook model and definitions of OML and IML.

a, Space-filling atomic models of a 66-subunit assembly based on our cryo-EM map. Two protofilaments in the 11-start direction are highlighted. Subunits on the outermost line (OML) are colored cyan and those on the innermost line (IML) are pink. The top figure shows a cross-sectional view from the tip of the hook. The bottom row shows three rotated side views with the base of the hook facing down. Scale bar, 50 Å. b, Polyhook model expanded by repeating 22-subunit segments (a subset from our 66-subunit model) and fitting them end-to-end 32 times. Both colored lines are 11-start helices on the supercoiled structure. The superhelical pitch P is 996 Å, the inner radius Ri of the superhelix is 45 Å and the outer radius Ro is 252 Å. The model dimensions agree well with our experimental data (Fig. 1a,b, pitch 1,001 Å). In its natural state, the hook contains ~130 subunits (Jones, C. J. et al. J. Mol. Biol. 212, 377–387, 1990), corresponding to a ~55 nm length (Aizawa, S.-I. The Flagellar World, Academic Press, 2014). Scale bar, 20 nm.

Supplementary Figure 4 Multiple sequence alignment of hinge regions.

The amino acid sequences of FlgE proteins from 20 bacterial species reveal shared hinge motifs. They are grouped by their flagellar position: peritrichous, polar and periplasmic (PP). Seventeen are Gram-negative and three are Gram-positive (Bacillus, Clostridium and Actinoplanes).

Supplementary Figure 5 D1–loop–D2 interface. Single protofilaments are shown at OML (left, cyan) and IML (right, pink).

Subunits on the same protofilament have the same conformation. The D1 loop is colored tan, and close-up views are shown in the insets. Although it appears that the D1 loop is close to the D2 domain from the next subunit (bottom right corners of insets), only a single polar interaction was identified. All other inter-atomic distances between these two domains are more than 3.6 Å apart.

Supplementary information

Supplementary Information

Supplementary Figs. 1–5 and Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Domain motions of the 11 protomers from one helical turn when aligned on D0. The animated figure was created with Pymol (Schrödinger).

Supplementary Video 2

Animation of the rotating hook, created by cyclically replacing the positions of the 11 protofilaments and morphing between their states. The movie was created with UCSF Chimera (Pettersen, E. F. et al. J. Comput. Chem. 25, 2004). First half: animation of full hook assembly. One protofilament is highlighted in orange. Second half: animation of a single protofilament.

Supplementary Video 3

Animated GIF of three classes from final 3D refinement. The superhelical structures from the three classes in the final multi-class 3D refinement iteration differed only slightly. Top row: Central slices through the 3D volume in orthogonal directions. Bottom row: Maximum intensity projections in the same directions. The spatial resolutions of these reconstructions were 3.3 Å, 3.3 Å and 3.0 Å. The subset of particle images from the reconstruction with the highest resolution was selected and refined further individually to obtain the final result at 2.9 Å resolution (Fig. 1). The images were created with cisTEM (Grant, T. et al. Elife 7, 2018).

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Shibata, S., Matsunami, H., Aizawa, SI. et al. Torque transmission mechanism of the curved bacterial flagellar hook revealed by cryo-EM. Nat Struct Mol Biol 26, 941–945 (2019). https://doi.org/10.1038/s41594-019-0301-3

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