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Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility

Nature Microbiology volume 6, pages 221–233 (2021)Cite this article

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Abstract

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

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Fig. 1: Both gliding motility and type 9 protein export require GldLM and the PMF.
Fig. 2: Structure of the F. johnsoniae GldLMʹ complex.
Fig. 3: Structure of the complete GldLM motor complex.
Fig. 4: Functional analysis of protonatable residues in the transmembrane domain of GldLM.
Fig. 5: The structural asymmetry of the GldLMʹ complex leads to important differences in residue environments between chains.
Fig. 6: Model for the mechanism of coupling proton flow to GldM rotation in the GldLM motor.

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Data availability

The cryo-EM volumes have been deposited in the Electron Microscopy Data Bank (EMDB) with accession codes EMD-10893 and EMD-10894, and the coordinates have been deposited in the Protein Data Bank (PDB) with accession code 6YS8. Source data are provided with the paper.

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Acknowledgements

We thank M. McBride and Y. Zhu for providing reagents for the genetic manipulation of F. johnsoniae. We thank L. Lavis for supplying the Janelia Fluor ligands, S. Hickman for advice on fluorescence imaging and A. Wainman for assistance with phase contrast imaging. We thank P. Guy for his involvement in producing the pulse-chase mCherry–CTD fusion construct and R. Berry for discussions. We acknowledge the use of the Central Oxford Structural Microscopy and Imaging Centre (COSMIC) and the Oxford Micron Advanced Imaging Facility. We thank K. Foster for providing additional imaging facilities. This work was supported by Wellcome Trust studentships to R.H.J. and A.S. (grant no. 1009136), a Biotechnology and Biological Sciences Research Council studentship to A.K. and Wellcome Trust Investigator Awards (grant nos. 107929/Z/15/Z and 100298/Z/12/Z). COSMIC was supported by a Wellcome Trust Collaborative Award (grant no. 201536/Z/16/Z), the Wolfson Foundation, a Royal Society Wolfson Refurbishment Grant, the John Fell Fund, and the EPA and Cephalosporin Trusts.

Author information

Author notes

  1. Felicity Alcock & Augustinas Silale

    Present address: CBCB, Newcastle University, Newcastle upon Tyne, UK

  2. These authors contributed equally: Rory Hennell James, Justin C. Deme.

Authors and Affiliations

  1. Sir William Dunn School of Pathology, University of Oxford, Oxford, UK

    Rory Hennell James, Justin C. Deme, Steven Johnson & Susan M. Lea

  2. Department of Biochemistry, University of Oxford, Oxford, UK

    Rory Hennell James, Justin C. Deme, Andreas Kjӕr, Felicity Alcock, Augustinas Silale, Frédéric Lauber & Ben C. Berks

  3. The Central Oxford Structural Molecular Imaging Centre (COSMIC), University of Oxford, Oxford, UK

    Justin C. Deme & Susan M. Lea

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Contributions

R.H.J. carried out all genetic and biochemical work except as credited otherwise. J.C.D. and S.M.L. collected electron microscopy data and determined the structures. A.K. developed and carried out the SprB tracking experiments including strain construction. F.A. carried out the pulse chase analysis of protein export. A.S. assayed cellular ATP levels. F.L. constructed the ∆gldL strain and produced the GldL and GldM antibodies. B.C.B. and S.M.L. conceived the project. All authors interpreted data and wrote the manuscript.

Corresponding authors

Correspondence to Ben C. Berks or Susan M. Lea.

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Additional information

Peer review information Nature Microbiology thanks Julien Bergeron, Dhana Gorasia and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Purification of the GldLM’ and PorLM complexes.

Coomassie Blue-stained SDS-PAGE gels and size-exclusion column traces of complexes used for cryoEM structure determination. In each case the size exclusion column fractions that were pooled to make the cryoEM grids are denoted by an arrow and correspond to the fractions analysed on the gels. A280 is the absorption at 280 nm in arbitrary units. a, GldLM225(produced from pRHJ007). b, GldLM232 (produced from pRHJ008). c, PorLM (produced from pRHJ006).

Extended Data Fig. 2 Cryo-EM workflow for GldLM′.

a, Exemplar micrograph collected at a defocus value of approximately −2.5 μm, Scale bar is 200 Å. b, Image processing workflow for GldLM’. Scale bar is 100 Å. c, Directional Fourier Shell Correlation (FSC) plot for GldLM′ calculated using the 3DFSC server52. d, Particle Orientation Distributions from final refined volume. Rotation (phi) is plotted along the x axis and tilt (theta) on the y axis. The area of the dot at the angular position is proportional to the number of particles assigned to that view.

Extended Data Fig. 3 Cryo-EM workflow for PorLM.

a, Image processing workflow for PorLM. Scale bar is 100 Å. b, Fourier Shell Correlation (FSC) plot for PorLM Red, phase randomised; Green, unmasked; Blue, masked; Black, corrected. c, Local resolution histogram of PorLM volume. d, Particle Orientation Distributions from final refined volume. Rotation (phi) is plotted along the x axis and tilt (theta) on the y axis. The area of the dot at the angular position is proportional to the number of particles assigned to that view.

Extended Data Fig. 4 Cryo-EM map quality and resolution estimates.

a, Fourier Shell Correlation (FSC) plot for the GldLM′ structure. The resolution at the gold-standard cutoff (FSC = 0.143) is indicated by the dashed line. Curves: Red, phase-randomized; Green, unmasked; blue, masked; black, MTF-corrected. b, Local resolution estimates (in Å) for the sharpened GldLM′ map. c, Representative modelled densities.

Extended Data Fig. 5

Cryo-EM data collection, refinement and validation statistics.

Extended Data Fig. 6 Contacts between the periplasmic loops of GldL and GldM.

The PDBePISA server (http://www.ebi.ac.uk/pdbe/pisart.html)60 was used to calculate the sizes of the interfaces between the periplasmic loops of each copy of GldL and the periplasmic domains of GldM. The periplasmic loops of GldL were taken to include residues 29–41.

Extended Data Fig. 7 Conservation analysis of the GldLM′ complex. a-c, Sequence conservation assessed using Consurf57.

a, The whole complex in cartoon representation. The chains shown individually in b and c are indicated with black arrows. b,c, GldM (chain B) and GldL (chain F) in surface representation. The left hand panels show the chains in the same orientation as the upper panel of a. d, Co-varying residue pairs in the first periplasmic domain of GldM identified using the Gremlin server61. The highest-scoring pairs (score cut-off 1.63) are shown on the structure of the GldLM′ complex. Contacts with a minimum atom-atom distance of <5 Å are shown in green, and 5–10 Å in yellow. Note, that no high-scoring GldM-GldM intermolecular pairs were observed for D1. Conversely dimerization of the other periplasmic domains is supported by co-variance with the following strongly co-varying pairs identified in the other domains D2:Val253-Ile284, Phe248-Glu-293, Tyr236-Arg261; D3: Thr327-Ala349, Asp331-Lys371; D4: Ala456-Asp483.

Extended Data Fig. 8 The full length PorLM volume is compatible with the shape of GldLM′ but not with the domain arrangements of earlier crystallographic structures of GldM (6ey4,) or PorM (6ey5) fragments.

The PorLM volume is contoured at 2.5 s and the upper and lower panels are related by a 90o rotation about the y axis. The approximate domain boundaries are indicated by the dashed lines and domains in these regions are labelled on the right hand side of the figure. The cryoEM structure of GldLM′ is positioned by docking the GldM′ TMHs onto the PorM TMHs as described in the text and Fig. 3. The D1 dimer of GldM 6ey415 is docked into the D1 portion of the volume and shows (1) poor fit within D1 because the centre of the closed crystallographic dimer protrudes from the volume and (2) poor fit in the D2-D4 region when no bend is introduced between D1-D2. PorM 6ey515 is positioned by placement of D2 into its portion of the volume and shows a poor fit for D3-4 which protrude from the volume. The hybrid model presented in Fig. 3 is shown for comparison, with GldM coloured as in other panels and GldL chains coloured individually.

Extended Data Fig. 9 Gliding motility and T9SS phenotypes of mutant strains.

Footnotes for table:1 Data from Fig. 4a.2 Activity of the major T9SS substrate chitinase in the extracellular medium. Includes data shown in Fig. 4b.3+ ++, spreads as well as the parental strain; ++ spreads less well than the parental strain; + residual spreading; - does not spread. Includes data shown in Fig. 4c. 4 Includes data from Supplementary Movie 1. Note that only strains possessing cell surface adhesins are able to adhere to glass slides, and that adhesin export to the cell surface requires the T9SS which in turn depends on functional GldLM. 5Data from Fig. 4d.

Extended Data Fig. 10 Closeup views of density around side chains discussed in manuscript.

Cryo-EM volumes contoured at 3 sigma. Key side chains are shown for both copies of GldM and for the GldL chain involved in the putative GldLE49 – GldMR9 salt-bridge.

Supplementary information

Supplementary Information

Supplementary Fig. 1, Supplementary Tables 1–3 and a list of Supplementary Videos 1–8.

Reporting Summary

Supplementary Video 1

Mutant strains gliding on glass. Wild-type, gldLY13A, gldLY13F, gldLK27A, gldLH30A, gldLE49D, gldMR9K, gldMY17A and gldMY17F cells are shown.

Supplementary Video 2

Fluorophore-labelled SprB adhesin moving on the surface of a wild-type cell.

Supplementary Video 3

Fluorophore-labelled SprB adhesin moving on the surface of a gldLY13A cell.

Supplementary Video 4

Fluorophore-labelled SprB adhesin moving on the surface of a gldLK27A cell.

Supplementary Video 5

Fluorophore-labelled SprB adhesin moving on the surface of a gldLH30A cell.

Supplementary Video 6

Fluorophore-labelled SprB adhesin moving on the surface of a gldMR9K cell.

Supplementary Video 7

Fluorophore-labelled SprB adhesin moving on the surface of a gldMY17F cell.

Supplementary Video 8

Animation of a rotary model of GldLM mechanism.

Source data

Source Data Fig. 1

Uncropped gels for Fig. 1.

Source Data Fig. 4

Uncropped gels for Fig. 4.

Source Data Fig. 4

Uncropped motility plates for Fig. 4.

Source Data Fig. 5

Uncropped gels for Fig. 5.

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Hennell James, R., Deme, J.C., Kjӕr, A. et al. Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility. Nat Microbiol 6, 221–233 (2021). https://doi.org/10.1038/s41564-020-00823-6

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