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A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM

Nature Structural & Molecular Biology volume 27, pages 589–597 (2020)Cite this article

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Abstract

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.

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Fig. 1: Cryo-EM structures of the Vo–VopQ complexes.
Fig. 2: VopQ is a membrane-inserted effector.
Fig. 3: Interaction between the Vo subcomplex and VopQ.
Fig. 4: VopQ is lethal to yeast with intact or partially assembled Vo subcomplexes.
Fig. 5: Working model for VopQ cytotoxicity.

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Data availability

The atomic coordinates of the Vo–VopQ complexes are deposited in the PDB with accession codes 6PE4 (Vo + 1 VopQ) and 6PE5 (Vo + 2 VopQ), respectively. The corresponding electron microscopy density maps are deposited in the Electron Microscopy Data Bank with accession codes EMD-20322 (Vo + 1 VopQ) and EMD-20323 (Vo + 2 VopQ), respectively. Source data for Fig. 2b, Extended Data Fig. 1b,d,e and Extended Data Fig. 6c–e are available with the paper online.

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Acknowledgements

We thank V. Tagliabracci, A. Sreelatha, X. Bai, X. Li and members of the Orth laboratory for discussions and editing. We thank X. Bai for technical assistance with cryo-EM image processing. We thank the Friedman laboratory for assistance with yeast cell disruption. We thank the Electron Microscopy Core Facility for assistance with negative staining grid preparation and image obtainment. We thank the Structural Biology Laboratory (SBL) for assistance with cryo-EM grid preparation and screening. Cryo-EM data were collected at the University of Texas Southwestern Medical Center Cryo-Electron Microscopy Facility, which is funded in part by the CPRIT Core Facility Support Award RP170644. We thank SBL and BioHPC for providing computational resources for cryo-EM data processing. This work was funded by the Welch Foundation grant I-1561 (to K.O.), Once Upon a Time… Foundation (to K.O.) and National Institutes of Health grant R01 GM115188 (to K.O.). K.O. is a W. W. Caruth, Jr, Biomedical Scholar with an Earl A. Forsythe Chair in Biomedical Science. D.R.T. is an Effie Marie Scholar.

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Authors and Affiliations

  1. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Wei Peng, Amanda K. Casey, Jessie Fernandez, Kelly A. Servage & Kim Orth

  2. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Wei Peng, Kelly A. Servage & Kim Orth

  3. Department of Microbiology, University of Georgia, Athens, GA, USA

    Emily M. Carpinone & Vincent J. Starai

  4. Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Zhe Chen, Yang Li & Diana R. Tomchick

  5. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Diana R. Tomchick & Kim Orth

  6. Department of Infectious Diseases, University of Georgia, Athens, GA, USA

    Vincent J. Starai

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Contributions

W.P. and K.O. conceived the project and designed the experiments. W.P., A.K.C., J.F., E.M.C. and K.A.S. conducted the experiments. Z.C., Y.L. and D.R.T. helped design experiments. V.J.S. helped design, interpret and perform experiments. All authors contributed to data analysis. W.P., A.K.C. and K.O. wrote the manuscript with input from all authors. K.O. supervized the project.

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Correspondence to Kim Orth.

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Extended data

Extended Data Fig. 1 Assembly of the Vo-VopQ complexes.

a,b,d, SEC analysis of the Vo-VopQ complexes with ribosomal protein contaminant eluted earlier (the first peak). c,d, Comparison of SEC profiles of the Vo-VopQ complexes and VopQ alone. Indicated fractions of Vo-VopQ complexes were pooled for cryo-EM sample preparation. e, Protein identification by mass spectrometry. Contaminating ribosomal proteins (indicated by a blue arrowhead in panel b), Vo subunits, and VopQ were confirmed. Only top-ranked proteins are listed to avoid over-interpretation or artificial bias. Uncropped gel images are shown in Source Data Extended Data Fig. 1.

Source data

Extended Data Fig. 2 Flowchart of cryo-EM data processing for the Vo-VopQ complexes.

Details are provided in online methods and in Extended Data Fig. 3.

Extended Data Fig. 3 Analysis of the Vo-VopQ complex cryo-EM data.

a, A representative cryo-EM micrograph. Scale bar = 2 nm. b, Representative 2D class averages. Scare bar = 50 Å. c, Angular distributions of particles used in the final 3D reconstructions, with the heights of the cylinders corresponding to the number of particles. d, EM density maps colored by local resolution. e, FSC curves for cross-validation between the models and the maps. Curves for the final refined models versus the reconstruction from total particles in black (sum), for the models refined against the reconstruction from only half of the particles versus the same reconstruction in blue (work), and for the same models versus the reconstruction from the other half of the particles in red (free).

Extended Data Fig. 4 Local density maps of the Vo-VopQ complexes.

The local density maps, shown as blue mesh, are contoured at 5.0 σ. Representative bulky residues are indicated. a, The electron density maps of representative elements from the complex of Vo + 1 VopQ. b, The EM density maps of the interacting elements from the Vo subcomplex and three VopQ molecules.

Extended Data Fig. 5 Structure of VopQ.

a, Comparison of the three VopQ molecules observed in the two Vo-VopQ complexes. b, The topology of VopQ. A cylinder cartoon indicates an α-helix and an arrow cartoon indicates a β-strand. Secondary structure elements of various domains are colored as in Fig. 2a. Residue boundaries are indicated for each individual secondary structure element.

Extended Data Fig. 6 Analysis of interaction between the Vo subcomplex and VopQ.

Interaction details between the Vo subcomplex and VopQ-2A and VopQ-2B. The figure scheme is as in Fig. 3b. b, Alignment of yeast and human Vo subunits a, c, and d. Identical residues are shaded blue and conserved residues are shaded yellow. Residues forming hydrogen bonds and salt bridges with VopQ are indicated by red triangles, either with main chain (empty triangles) or side chain (solid triangles) atoms. Secondary structural elements of subunit c are indicated at the bottom. c, Pulldown assays to test interactions between the Vo subcomplex and VopQ variants. Two bands corresponding to subunit a are observed due to protein degradation. Panel c (left), the Vo subcomplex was incubated with the affinity resin as bait to pull down VopQ variants. Panel c (right), non-specific binding of ClyA to the affinity resin, which was used for blocking the affinity resin in the left hand panel. d,e, Western Blot (anti-Flag) showing the expression of VopQ variants (indicated by arrow) in the yeast growth inhibition assay. f, Comparison of the Vo subcomplexes from the structures of the Vo subcomplex alone (PDB 6C6L), Vo + 1 VopQ, and Vo + 2 VopQ. The c-ring of the Vo + 1 VopQ or the Vo + 2 VopQ complex is aligned over the c-ring of the Vo subcomplex alone. The orange arrow indicates the slight movement of subunits a and d due to binding of 2 VopQ molecules in the Vo + 2 VopQ complex. Uncropped gel and blot images are shown in Source Data Extended Data Fig. 6.

Source data

Extended Data Fig. 7 VopQ may block V-ATPase assembly and possibly binds to V1Vo complex in state 2.

a, Vo + 1 VopQ or Vo + 2 VopQ is aligned with V1Vo (PDB 5VOY) over Vo subunit a, one of the stator components. Color scheme: V-ATPase subunits a (green), V1_C (cyan), V1_E (black), and V1_G (red); all other subunits are colored grey. VopQ molecules are indicated. Only VopQ molecules in Vo + 1 VopQ and Vo + 2 VopQ are shown for simplicity. b-d, The complexes of Vo + 1 VopQ and Vo + 2 VopQ are aligned over subunit d in the V1Vo complexes in state 1 (PDB 5VOX), state 2 (PDB 5VOY), and state 3 (PDB 5VOZ).

Extended Data Fig. 8 A potential fourth binding site for VopQ within the Vo subcomplex.

a, Sequence alignment of yeast Vo subunits c, c′, and c″, prepared as in Extended Data Fig. 6b. The three subunits share sequence homology (51.2% identity and 84.5% similarity for c and c′, 29.1% identity and 66.9% similarity for c and homologous region of c″). b, Rotation of the c-ring (Vo + 2 VopQ) and clash analysis between Vo subunits and VopQ. Cα atoms of c″_T97 and c(5)_I54 are shown as green spheres. The rotation was achieved by aligning subunit c(8) in Vo + 2 VopQ complex with various c-ring subunits in Vo + 1 VopQ complex. VopQ-2A and VopQ-2B are used for validation, while VopQ-1 is kept stationary and used for clash analysis. Four sites shown here (occupied by the purple VopQ) allow VopQ to bind based on clash analysis. The one revealed by rotation of 8 × 36° is not experimentally observed since it spatially clashes with subunit a in the intact Vo subcomplex, indicated by a red circle. c, Four binding sites for VopQ within the Vo subcomplex. Three are observed in the structures and the fourth, VopQ3, is predicted based on structure analysis.

Extended Data Fig. 9 The closed and open states of c-ring glutamates for proton transport.

a, Closed or open states of glutamates in crystal structures of various ATPase c-rings: Ilyobacter tartaricus F-ATPase c11-ring (2.4 Å, PDB 1YCE), Spirulina platensis F-ATPase c15-ring (2.1 Å, PDB 2WIE), Saccharomyces cerevisiae F-ATPase c10-ring in complex with oligomycin (1.9 Å, PDB 4F4S), Enterococcus hirae V-ATPase c10-ring (2.1 Å, PDB 2BL2). Open states of yeast F-ATPase c-ring glutamate sides chains are caused by crystallization reagent 2-methyl-2,4-pentanediol and closed states are restored by the inhibitor oligomycin. b, Closed and open states of the Vo c-ring glutamates in the structures of the Vo subcomplex alone (PDB 6C6L), Vo + 1 VopQ, and Vo + 2 VopQ. Representative local density maps for glutamates are shown as blue mesh at the contour level of 5.0 σ.

Extended Data Fig. 10 Unmodeled densities in the Vo-VopQ complexes.

a, Unmodeled densities shown at the contour level of 5.0 σ. b, Unmodeled densities shown at the contour level of 3.0 σ. E137 of subunit c(1), shown as a stick, is in proximity to lipid density ②. Relative levels of lipids, either outside or inside of the c-ring, are indicated by the bifurcation points for two hydrophobic hydrocarbon chains of lipid molecules. ‘?’ indicates an unknown density and ‘*’ indicates a possible yeast sterol density. c, Electrostatic surface potential of Vo +1 VopQ complex (left) and the inner surface of the c-ring (right). This figure scheme is as in Fig. 2c.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2. List of yeast strains and plasmids used in this study.

Reporting Summary

Source data

Source Data Fig. 2

Unprocessed gel images.

Source Data Extended Data Fig. 1

Unprocessed gel images.

Source Data Extended Data Fig. 6

Unprocessed western blots and gel images.

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Peng, W., Casey, A.K., Fernandez, J. et al. A distinct inhibitory mechanism of the V-ATPase by Vibrio VopQ revealed by cryo-EM. Nat Struct Mol Biol 27, 589–597 (2020). https://doi.org/10.1038/s41594-020-0429-1

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  • DOI: https://doi.org/10.1038/s41594-020-0429-1

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