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The structure of the periplasmic FlaG–FlaF complex and its essential role for archaellar swimming motility

Abstract

Motility structures are vital in all three domains of life. In Archaea, motility is mediated by the archaellum, a rotating type IV pilus-like structure that is a unique nanomachine for swimming motility in nature. Whereas periplasmic FlaF binds the surface layer (S-layer), the structure, assembly and roles of other periplasmic components remain enigmatic, limiting our knowledge of the archaellum’s functional interactions. Here, we find that the periplasmic protein FlaG and the association with its paralogue FlaF are essential for archaellation and motility. Therefore, we determine the crystal structure of Sulfolobus acidocaldarius soluble FlaG (sFlaG), which reveals a β-sandwich fold resembling the S-layer-interacting FlaF soluble domain (sFlaF). Furthermore, we solve the sFlaG2–sFlaF2 co-crystal structure, define its heterotetrameric complex in solution by small-angle X-ray scattering and find that mutations that disrupt the complex abolish motility. Interestingly, the sFlaF and sFlaG of Pyrococcus furiosus form a globular complex, whereas sFlaG alone forms a filament, indicating that FlaF can regulate FlaG filament assembly. Strikingly, Sulfolobus cells that lack the S-layer component bound by FlaF assemble archaella but cannot swim. These collective results support a model where a FlaG filament capped by a FlaG–FlaF complex anchors the archaellum to the S-layer to allow motility.

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Fig. 1: The sFlaG crystal structure exhibits a β-sandwich fold that is stable in pH 3 buffer solution as shown by SAXS.
Fig. 2: sFlaG interacts with sFlaF to form a heterotetrameric complex.
Fig. 3: Mutations that disrupt heterotetrameric complex formation by sFlaG and sFlaF also abolish swimming motility in S. acidocaldarius.
Fig. 4: Both FlaF and FlaG are found in the periplasm, and maturation of FlaG N-terminal cleavage is essential for cell motility.
Fig. 5: S-layer protein SlaA deletion cells impair cell motility while maintaining archaella in S. islandicus.
Fig. 6: Pfu sFlaF limits Pfu sFlaG filament formation that results in our working model.

Data availability

The X-ray diffraction data and coordinates of the sFlaG and sFlaG–sFlaF complex structures have been deposited with the PDB: 5TUH (sFlaG); 5TUG (sFlaGWT/sFlaFWT); and 6PBK (sFlaGV118K/sFlaFWT). The SAXS data have been deposited with the SASBDB: SASDEU7 (sFlaG); SASDES7 (sFlaGWT/sFlaFWT); SASDEV7 (sFlaGWT/sFlaFI96Y); and SASDET7 (sFlaGV118K/sFlaFWT). Source data for Fig. 4 and Supplementary Figs. 5, 7, 10 and 11 are provided with the paper.

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Acknowledgements

P.T. received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement no. 686647. S.-V.A. was funded by the Deutsche Forschungsgemeinschaft (German Research Foundation) under project no. 403222702-SFB 1381. The work was conducted at ALS, a national user facility operated by the Lawrence Berkeley National Laboratory on behalf of the US Department of Energy’s Office of Basic Energy Sciences, through the Integrated Diffraction Analysis Technologies program, supported by US Department of Energy Office of Biological and Environmental Research. Additional support comes from the National Institutes of Health (NIH) grant no. P30GM124169. J.A.T. acknowledges start-up funds from the University of Texas Safety Tracking and Reporting System (NIH grant no. R35CA220430) and the Robert A. Welch Chair in Chemistry (grant no. CFS127800-80-101399-50). C.Z., R.L.W. and R.J.W. are supported by a grant from NASA (National Aeronautics and Space Administration) through the NASA Astrobiology Institute under cooperative agreement no. NNA13AA91A, issued through the Science Mission Directorate. We thank S. Robinson from the Microscopy Suite at the Beckman Institute for Advance Science and Technology, University of Illinois at Urbana-Champaign, for providing TEM assistance. We especially thank K. Burnett for her assistance on the SAXS data collection of the sFlaG and sFlaG–sFlaF complexes, G. Hura for the SAXS similarity calculation suggestion and S. Classen for his assistance on the X-ray crystallography data collection at the SIBYLS Beamline (BL12.3.1). We thank A. Ghosh for technical help; B. Tutt at the Department of Scientific Publications at the MD Anderson Cancer Center for proofreading and editing our manuscript; and we thank P. Simpson for technical assistance with imaging the P. furiosus FlaF and FlaG assemblies. The TEM is operated by the University of Freiburg, Faculty of Biology, as a partner unit within the Microscopy and Image Analysis Platform, Freiburg.

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Contributions

C.-L.T, P.T., J.A.T. and S.-V.A. designed the experiments, analysed the data and wrote the manuscript. C.-L.T. and J.A.T. contributed to protein crystallization, SAXS, X-ray data collection, structure determination, data analyses, the initial EM model reconstruction and the figures. P.T., S.S., M.R.-F., A.B., P.C., M.B. and S.-V.A. contributed to protein production and purification, SEC and reconstitution, microscale thermophoresis, cell motility, cell fractionation, data analysis, TEM analysis and the figures. C.Z., R.L.W. and R.J.W. contributed to the S-layer protein knockout, TEM analysis and the figures.

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Correspondence to John A. Tainer or Sonja-Verena Albers.

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Supplementary Information

Supplementary Results, Supplementary Figs. 1–11, Supplementary Tables 1–3 and Supplementary References.

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Supplementary Data 3

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Tsai, CL., Tripp, P., Sivabalasarma, S. et al. The structure of the periplasmic FlaG–FlaF complex and its essential role for archaellar swimming motility. Nat Microbiol 5, 216–225 (2020). https://doi.org/10.1038/s41564-019-0622-3

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