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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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.
McCarter, L. The multiple identities of Vibrio parahaemolyticus. J. Mol. Microbiol. Biotechnol. 1, 51–57 (1999).
Tran, L. et al. Determination of the infectious nature of the agent of acute hepatopancreatic necrosis syndrome affecting penaeid shrimp. Dis. Aquat. Organ. 105, 45–55 (2013).
Lai, H. C. et al. Pathogenesis of acute hepatopancreatic necrosis disease (AHPND) in shrimp. Fish Shellfish Immunol. 47, 1006–1014 (2015).
Santos, M.D., Salomon, D., Li, P., Krachler, A.M. & Orth, K. in Comprehensive Sourcebook of Bacterial Protein Toxins 4th edn (eds Alouf, J. et al.) 230–260 (Elevier, 2015).
Zhang, L. & Orth, K. Virulence determinants for Vibrio parahaemolyticus infection. Curr. Opin. Microbiol. 16, 70–77 (2013).
Burdette, D. L., Yarbrough, M. L., Orvedahl, A., Gilpin, C. J. & Orth, K. Vibrio parahaemolyticus orchestrates a multifaceted host cell infection by induction of autophagy, cell rounding, and then cell lysis. Proc. Natl Acad. Sci. USA 105, 12497–12502 (2008).
Burdette, D. L., Seemann, J. & Orth, K. Vibrio VopQ induces PI3-kinase-independent autophagy and antagonizes phagocytosis. Mol. Microbiol. 73, 639–649 (2009).
Yarbrough, M. L. et al. AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream signaling. Science 323, 269–272 (2009).
Broberg, C. A., Zhang, L., Gonzalez, H., Laskowski-Arce, M. A. & Orth, K. A Vibrio effector protein is an inositol phosphatase and disrupts host cell membrane integrity. Science 329, 1660–1662 (2010).
Salomon, D. et al. Effectors of animal and plant pathogens use a common domain to bind host phosphoinositides. Nat. Commun. 4, 2973 (2013).
Sreelatha, A. et al. Vibrio effector protein, VopQ, forms a lysosomal gated channel that disrupts host ion homeostasis and autophagic flux. Proc. Natl Acad. Sci. USA 110, 11559–11564 (2013).
Higa, N. et al. Vibrio parahaemolyticus effector proteins suppress inflammasome activation by interfering with host autophagy signaling. PLoS Pathog. 9, e1003142 (2013).
Sreelatha, A. et al. Vibrio effector protein VopQ inhibits fusion of V-ATPase-containing membranes. Proc. Natl Acad. Sci. USA 112, 100–105 (2015).
Matsuda, S., Okada, N., Kodama, T., Honda, T. & Iida, T. A cytotoxic type III secretion effector of Vibrio parahaemolyticus targets vacuolar H+-ATPase subunit c and ruptures host cell lysosomes. PLoS Pathog. 8, e1002803 (2012).
Forgac, M. Vacuolar ATPases: rotary proton pumps in physiology and pathophysiology. Nat. Rev. Mol. Cell Biol. 8, 917–929 (2007).
Cotter, K., Stransky, L., McGuire, C. & Forgac, M. Recent insights into the structure, regulation, and function of the V-ATPases. Trends Biochem. Sci. 40, 611–622 (2015).
Oot, R. A., Couoh-Cardel, S., Sharma, S., Stam, N. J. & Wilkens, S. Breaking up and making up: the secret life of the vacuolar H+-ATPase. Protein Sci. 26, 896–909 (2017).
Zoncu, R. et al. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H+-ATPase. Science 334, 678–683 (2011).
Xu, Y. et al. A bacterial effector reveals the V-ATPase-ATG16L1 axis that initiates xenophagy. Cell 178, 552–566.e20 (2019).
Xu, L. et al. Inhibition of host vacuolar H+-ATPase activity by a Legionella pneumophila effector. PLoS Pathog. 6, e1000822 (2010).
Kane, P. M. Disassembly and reassembly of the yeast vacuolar H+-ATPase in vivo. J. Biol. Chem. 270, 17025–17032 (1995).
Mazhab-Jafari, M. T. et al. Atomic model for the membrane-embedded VO motor of a eukaryotic V-ATPase. Nature 539, 118–122 (2016).
Roh, S. H. et al. The 3.5-Å cryoEM structure of nanodisc-reconstituted yeast vacuolar ATPase VO proton channel. Mol. Cell 69, 993–1004 (2018).
Vasanthakumar, T. et al. Structural comparison of the vacuolar and Golgi V-ATPases from Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA 116, 7272–7277 (2019).
PyMOL Molecular Graphics System v.1.8 (Schrödinger, 2015).
Laskowski, R. A., Jablonska, J., Pravda, L., Varekova, R. S. & Thornton, J. M. PDBsum: structural summaries of PDB entries. Protein Sci. 27, 129–134 (2018).
Holm, L. & Sander, C. Protein structure comparison by alignment of distance matrices. J. Mol. Biol. 233, 123–138 (1993).
Iwabuchi, N. et al. Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma. Biochem. Biophys. Res. Commun. 513, 952–957 (2019).
Peng, W. et al. High-resolution cryo-EM structures of the E. coli hemolysin ClyA oligomers. PLoS ONE 14, e0213423 (2019).
Strop, P. & Brunger, A. T. Refractive index-based determination of detergent concentration and its application to the study of membrane proteins. Protein Sci. 14, 2207–2211 (2005).
Dal Peraro, M. & van der Goot, F. G. Pore-forming toxins: ancient, but never really out of fashion. Nat. Rev. Microbiol. 14, 77–92 (2016).
Malkus, P., Graham, L. A., Stevens, T. H. & Schekman, R. Role of Vma21p in assembly and transport of the yeast vacuolar ATPase. Mol. Biol. Cell 15, 5075–5091 (2004).
Graham, L. A., Flannery, A. R. & Stevens, T. H. Structure and assembly of the yeast V-ATPase. J. Bioenerg. Biomembr. 35, 301–312 (2003).
Mazhab-Jafari, M. T. & Rubinstein, J. L. Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases. Sci. Adv. 2, e1600725 (2016).
Zhao, J. et al. Molecular basis for the binding and modulation of V-ATPase by a bacterial effector protein. PLoS Pathog. 13, e1006394 (2017).
Zhao, J., Benlekbir, S. & Rubinstein, J. L. Electron cryomicroscopy observation of rotational states in a eukaryotic V-ATPase. Nature 521, 241–245 (2015).
Muller, V. & Gruber, G. ATP synthases: structure, function and evolution of unique energy converters. Cell. Mol. Life Sci. 60, 474–494 (2003).
Kuhlbrandt, W. Structure and mechanisms of F-type ATP synthases. Annu. Rev. Biochem. 88, 515–549 (2019).
Meier, T., Polzer, P., Diederichs, K., Welte, W. & Dimroth, P. Structure of the rotor ring of F-type Na+-ATPase from Ilyobacter tartaricus. Science 308, 659–662 (2005).
Pogoryelov, D., Yildiz, O., Faraldo-Gomez, J. D. & Meier, T. High-resolution structure of the rotor ring of a proton-dependent ATP synthase. Nat. Struct. Mol. Biol. 16, 1068–1073 (2009).
Preiss, L., Yildiz, O., Hicks, D. B., Krulwich, T. A. & Meier, T. A new type of proton coordination in an F1Fo-ATP synthase rotor ring. PLoS Biol. 8, e1000443 (2010).
Pogoryelov, D. et al. Microscopic rotary mechanism of ion translocation in the Fo complex of ATP synthases. Nat. Chem. Biol. 6, 891–899 (2010).
Symersky, J. et al. Structure of the c10 ring of the yeast mitochondrial ATP synthase in the open conformation. Nat. Struct. Mol. Biol. 19, 485–491 (2012).
Symersky, J., Osowski, D., Walters, D. E. & Mueller, D. M. Oligomycin frames a common drug-binding site in the ATP synthase. Proc. Natl Acad. Sci. USA 109, 13961–13965 (2012).
Murata, T., Yamato, I., Kakinuma, Y., Leslie, A. G. & Walker, J. E. Structure of the rotor of the V-type Na+-ATPase from Enterococcus hirae. Science 308, 654–659 (2005).
Zinser, E., Paltauf, F. & Daum, G. Sterol composition of yeast organelle membranes and subcellular distribution of enzymes involved in sterol metabolism. J. Bacteriol. 175, 2853–2858 (1993).
Zhang, Y. Q. et al. Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs. PLoS Pathog. 6, e1000939 (2010).
Breton, S. & Brown, D. Regulation of luminal acidification by the V-ATPase. Physiology 28, 318–329 (2013).
Pérez-Sayáns, M., Somoza-Martin, J. M., Barros-Angueira, F., Rey, J. M. & García-García, A. V-ATPase inhibitors and implication in cancer treatment. Cancer Treat. Rev. 35, 707–713 (2009).
Hernandez, A., Serrano-Bueno, G., Perez-Castineira, J. R. & Serrano, A. Intracellular proton pumps as targets in chemotherapy: V-ATPases and cancer. Curr. Pharm. Des. 18, 1383–1394 (2012).
Wiedmann, R. M. et al. The V-ATPase-inhibitor archazolid abrogates tumor metastasis via inhibition of endocytic activation of the Rho-GTPase Rac1. Cancer Res. 72, 5976–5987 (2012).
Merk, H. et al. Inhibition of the V-ATPase by archazolid A: a new strategy to inhibit EMT. Mol. Cancer Ther. 16, 2329–2339 (2017).
Couoh-Cardel, S., Milgrom, E. & Wilkens, S. Affinity purification and structural features of the yeast vacuolar ATPase Vo membrane sector. J. Biol. Chem. 290, 27959–27971 (2015).
Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).
Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. Elife 5, e18722 (2016).
Zivanov, J. et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Elife 7, e42166 (2018).
Morin, A. et al. Collaboration gets the most out of software. Elife 2, e01456 (2013).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).
Amunts, A. et al. Structure of the yeast mitochondrial large ribosomal subunit. Science 343, 1485–1489 (2014).
DiMaio, F., Zhang, J., Chiu, W. & Baker, D. Cryo-EM model validation using independent map reconstructions. Protein Sci. 22, 865–868 (2013).
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).
Pettersen, E. F. et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324 (2014).
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.
The authors declare no competing interests.
Peer review information Beth Moorefield 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.
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.
Details are provided in online methods and in Extended Data Fig. 3.
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).
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.
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.
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.
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).
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.
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.
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.
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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