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Na+-V-ATPase inhibitor curbs VRE growth and unveils Na+ pathway structure

Nature Structural & Molecular Biology volume 32, pages 450–458 (2025)Cite this article

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Abstract

Vancomycin-resistant Enterococcus faecium (VRE) is a major cause of nosocomial infections, particularly endocarditis and sepsis. With the diminishing effectiveness of antibiotics against VRE, new antimicrobial agents are urgently needed. Our previous research demonstrated the crucial role of Na+-transporting V-ATPase in Enterococcus hirae for growth under alkaline conditions. In this study, we identified a compound, V-161, from 70,600 compounds, which markedly inhibits E. hirae V-ATPase activity. V-161 not only inhibits VRE growth in alkaline conditions but also significantly suppresses VRE colonization in the mouse small intestine. Furthermore, we unveiled the high-resolution structure of the membrane VO part due to V-161 binding. V-161 binds to the interface of the c-ring and a-subunit, constituting the Na+ transport pathway in the membrane, thereby halting its rotation. This structural insight presents potential avenues for developing therapeutic agents for VRE treatment and elucidates the Na+ transport pathway and mechanism.

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Fig. 1: Identification and characterization of compound V-161.
Fig. 2: Inhibitory effects of V-161 on growth in vitro and in vivo.
Fig. 3: Structure of the VO part of EhV-ATPase with V-161.
Fig. 4: Na+ transport pathway of EhV-ATPase.
Fig. 5: Proposed mechanisms of Na+ transport and inhibition in EhV-ATPase.

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Data availability

Cryo-EM maps are deposited in the Electron Microscopy Data Bank (EMDB) under accession code EMD-37440 for maps of the VO part of EhV-ATPase from focused refinement. Atomic models are deposited in the Protein Data Bank (PDB) under accession code PDB 8WCI. Source data are provided with this paper.

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Acknowledgements

This paper is dedicated to the late Y. Kakinuma, who passed away at the age of 64 in January 2016, in memory of his important contributions to the research of EhV-ATPase. We thank the staff at the KEK Structure Biology Research Center and the University of Tokyo for assistance with cryo-EM operation. We also thank K. Mizutani, T. Ajiro and I. Seiki for their technical support with the in vitro experiments. This work was supported by Grant-in-Aid for Scientific Research, from Japan Society for the Promotion of Science (JSPS) under grant numbers 18H05425 (T. Murata), 21H02409 (T. Murata), 24H00550 (T. Murata), 23H02427 (T. Moriya), 18H05424 (R.I.), 21H02454 (R.I.), 21K15060 (A.O.), 18H05426 (M.I.), 21K06108 (S.Y.), 19H03465 (Y.G.) and 23K18285 (Y.G.); by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from Japan Agency for Medical Research and Development (AMED) under grant numbers JP23ama121001 (T.S.), JP23ama121002 (M.K.), JP23ama121007 (S.I.), JP23ama121013 (T. Murata), JP23ama121023 (M.I.), JP23ama121053 (H.K.), 21fk0108092h0003 (T. Murata), 23fk0108604h0903 (H.T.) and 23fk0108665h0301 (H.T.) and JP223fa627003 (Y.G.); by Research Program on ensuring Food Safety from Japanese Ministry of Health, Labour and Welfare under grant numbers 21KA1004 (H.T.); by Ministry of Education, Culture, Sports, Science and Technology (MEXT) under grant numbers 22K07067 (H.T.), 22K16368 (Y.H.) and 22K07052 (J.K.); by the grant of OML Project by the National Institutes of Natural Sciences (NINS program No. OML022301); by JST FOREST Program under grant number JPMJFR225D (Y.G.); and by IAAR Research Support Program and Initiative for Realizing Diversity in the Research Environment (T. Murata, K. Suzuki), Chiba University, Japan.

Author information

Author notes

  1. These authors contributed equally: Kano Suzuki, Yoshiyuki Goto.

Authors and Affiliations

  1. Department of Chemistry, Graduate School of Science, Chiba University, Chiba, Japan

    Kano Suzuki, Kouki Shimizu, Shohei Abe, Katsuhiko Moriyama, Satoshi Yasuda, Sho Mikuriya, Fabiana L. Imai, Satoshi Ogasawara & Takeshi Murata

  2. Department of Quantum Life Science, Graduate School of Science, Chiba University, Chiba, Japan

    Kano Suzuki, Katsuhiko Moriyama, Satoshi Yasuda, Satoshi Ogasawara & Takeshi Murata

  3. Membrane Protein Research Center, Chiba University, Chiba, Japan

    Kano Suzuki, Yoshiyuki Goto, Katsuhiko Moriyama, Satoshi Yasuda, Satoshi Ogasawara & Takeshi Murata

  4. Division of Molecular Immunology, Medical Mycology Research Center, Chiba University, Chiba, Japan

    Yoshiyuki Goto

  5. Division of Pandemic and Post-disaster Infectious Diseases, Research Institute of Disaster Medicine, Chiba University, Chiba, Japan

    Yoshiyuki Goto

  6. Division of Infectious Disease Vaccine R&D, Research Institute of Disaster Medicine, Chiba University, Chiba, Japan

    Yoshiyuki Goto

  7. Synergy Institute for Futuristic Mucosal Vaccine Research and Development (cSIMVa), Chiba University, Chiba, Japan

    Yoshiyuki Goto

  8. Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki, Japan

    Akihiro Otomo & Ryota Iino

  9. The Graduate Institute for Advanced Studies, SOKENDAI, Shonan Village, Hayama, Japan

    Akihiro Otomo & Ryota Iino

  10. Department of Bacteriology, Graduate School of Medicine, Gunma University, Maebashi, Japan

    Yusuke Hashimoto & Haruyoshi Tomita

  11. Laboratory of Bacterial Drug Resistance, Graduate School of Medicine, Gunma University, Maebashi, Japan

    Jun Kurushima & Haruyoshi Tomita

  12. Structural Biology Research Center, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Japan

    Naruhiko Adachi, Masato Kawasaki, Toshiya Senda & Toshio Moriya

  13. Life Science Center for Survival Dynamics, Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan

    Naruhiko Adachi

  14. Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Yumi Sato & So Iwata

  15. School of High Energy Accelerator Science, SOKENDAI, Tsukuba, Japan

    Toshiya Senda

  16. Faculty of Pure and Applied Sciences, University of Tsukuba, Tsukuba, Japan

    Toshiya Senda

  17. Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan

    Mitsunori Ikeguchi & Takeshi Murata

Authors
  1. Kano Suzuki
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  2. Yoshiyuki Goto
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  22. Takeshi Murata
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Contributions

T. Murata conceived the project. K. Suzuki, A.O., K. Shimizu, S.M., F.L.I. and T. Murata prepared the protein samples. K. Shimizu, F.L.I., Y.S. and S.I. screened the inhibitors. K.M. synthesized the inhibitor. A.O. and R.I. performed single-molecule rotation experiments. Y.H., J.K. and H.T. developed deficient strains. K. Suzuki, A.O., K. Shimizu, S.A., S.M., F.L.I. and Y.S. performed the in vitro experiments. Y.G., K. Shimizu, S.A. and S.O. performed in vivo experiments. K. Suzuki, S.M., N.A., M.K., T.S. and T. Moriya performed cryo-EM data collection and processing. K. Suzuki built and refined the atomic models. S.Y. and M.I. performed MD simulations. K. Suzuki, Y.G. A.O., K.M., S.Y., R.I., T. Moriya and T. Murata analyzed the results, prepared the figures and wrote the paper. All the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Takeshi Murata.

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The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Alastair Stewart, Stephan Wilkens and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editors: Katarzyna Ciazynska and Dimitris Typas, in collaboration with the Nature Structural & Molecular Biology team.

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Extended data

Extended Data Fig. 1 Previous structural analyses of Na+-transporting V-ATPase (EhV-ATPase) from Enterococcus hirae.

(a) Mechanistic model of EhV-ATPase. (b) Crystal structure (PDB ID: 3VR4) of the EhV1-ATPase (A3B3DF complex)10. (c) Crystal structure (PDB ID: 2BL2) of the Na+-bound c-ring20. The right panel displays a magnified view of the Na+-binding site. (d) Local resolution assessment of the reconstructed map from a previously reported Cryo-EM structure of EhV-ATPase36.

Extended Data Fig. 2 Na+-transporting V-ATPase is required for VRE growth under alkaline conditions and for colonization in the small intestine in Mice.

(a) The ntpI gene cording the a-subunit was replaced with the cat gene to generate the a-subunit-deficient VRE (ΔEfV). Refer to Supplementary Methods. (b) Susceptibility of ΔEfV to the antibiotics was comparable to the WT VRE. (c) Time-course growth curves of WT and ΔEfV under varying pH conditions. (d) Growth comparison of WT and ΔEfV at 12 h, as indicated by the red line in (c). (c–d) All data are presented as mean ± standard error of the mean from three independent experiments. P values were determined using a two-tailed t-test. (e) The number of VRE in the intestinal contents and faeces of WT or ΔEfV-infected mice (n = 5 per group). P values, determined using a two-tailed Mann-Whitney U test, are shown in parentheses. L.o.D., limit of detection. Statistical significance is denoted as *P < 0.05, **P < 0.01. NS indicates no significant difference.

Source data

Extended Data Fig. 3 Cryo-EM data processing and analysis focusing on the VO part.

(a) Workflow diagram illustrating the process to derive the final VO part Cryo-EM map. 3,264 micrographs were collected and processed for the cryo-EM reconstruction. (b) Fourier shell correlation (FSC) curves for the reconstructed Cryo-EM half-set maps and between the atomic model and full-set map. (c) Angular distribution plot. (d) Local resolution of the reconstructed map. The right panel provides a detailed view of the V-161 binding site at the interface of the c-ring and the a-subunit.

Extended Data Fig. 4 Example model-in-map fit for various helices, cardiolipin, and V-161 map regions.

(a) Overall structure of the VO part showing positions (b–k) of helices. (b–n) Model-in-map fits for: (b,c) Na+-bound c-subunit, (d,e) Na+-unbound c-subunit situated at the centre region of the a-subunit boundary (black), (f–k) C-terminal domain of a-subunit, (l) Cardiolipin, and (m,n) V-161. The surface structures of the Cryo-EM densities are rendered at 5.0 sigma (b–m) and 9.0 sigma (n) are shown, respectively.

Extended Data Fig. 5 Na+-binding sites in the VO part.

(a) Model-in-map of the VO part presented in a cross-sectional top view as in Fig. 3e. The left panel shows a magnified view of the empty Na+-binding site, as highlighted in the red box. (b–k) Model-in-maps of all Na+-binding sites, corresponding to the black boxes in (a). (l) The Na+-binding site in (b) is superimposed at all Cα atom onto those of 8 Na+ bound sites. (m) The Na+-binding site in (b) is superimposed at Cα atom onto that of the empty Na+-binding site (c) shown in black. The surface structures of the Cryo-EM densities are rendered at 5.0 sigma are shown.

Extended Data Fig. 6 Schematic representation of the interactions between the ligands and VO part.

(a–c) The interactions between the ligands (a: V-161, b: Na+, c: cardiolipin) and VO part were analysed using the Ligplot+ program67. Hydrogen bonds are shown as green dashed lines. Van der Waals contacts are represented by the spoked arcs oriented toward the ligand atoms they contact. The range of distances defining van derWaals contacts is 2.9–3.9 Å.

Extended Data Fig. 7 Comparison of V-161 binding site of EhV-ATPase with the corresponding sites of other V- and F-ATPases.

(a–d) Detailed views of the V-161 binding site of EhV-ATPase (a,b) and the corresponding site of F-ATPase (PDB ID: 6RDC) from Polytomella sp. Pringsheim (c,d), viewed as in Figs. 4i, j. (e) Sequence alignment of the c-subunit and the a-subunit from various V-ATPases (Na+-transporting types: from E. hirae and E. faecium; H+-transporting types: from rat and yeast) and F-ATPases (Na+-transporting types: from I. tartaricus and P. modestum; H+-transporting types: from bovine mitochondria (PDB ID: 6ZQM), yeast mitochondria (PDB ID: 6B8H), and Polytomella sp. Pringsheim (PDB ID: 6RDC)). Box colours adhere to the scheme in Fig. 3g. Residue numbers for E. hirae and Polytomella sp. Pringsheim are provided above and below the alignment, respectively. The residues c-T140 and a-N615 of Na+-transporting V-ATPase (shown in red boxes) are not conserved in other ATPases.

Extended Data Fig. 8 Comparison of ion transport pathways of Na+-transporting EhV-ATPase and H+-transporting V-ATPases.

The colours of the ion transport pathways are consistent with those in Fig. 4. (a–f) Side views (as in Fig. 4j) and top views (as in Fig. 4c) of the electronegative cavity leading to the periplasm of the VO parts from E. hirae (a, d), yeast (b, e; PDB ID: 6M0S), and rat (c, f; PDB ID: 6VQC). Residues conserved across both the Na+- and H+-transporting V-ATPases are highlighted in cyan boxes. The V-161 binding sites specific to EhV-ATPase and analogous sites in H+-transporting V-ATPases from yeast and rat are shown in red boxes.

Supplementary information

Supplementary Information

Supplementary Tables 1–2, Supplementary Figs. 1–3, Supplementary Methods

Reporting Summary

Supplementary Video 1

Structure of the VO part of EhV-ATPase with V-161.

Supplementary Video 2

Na+ transport pathway of EhV-ATPase.

Supplementary Video 3

Na+ transport pathway to periplasm predicted using MD simulations.

Supplementary Video 4

Proposed mechanisms of Na+ transport and inhibition in EhV-ATPase.

Supplementary Data 1

Source data for Supplementary Fig. 1.

Source data

Source Data Fig. 1

Statistical Source Data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 4

Statistical Source Data

Source Data Extended Data Fig./Table 2

Unprocessed gels related to Extended Data Fig. 2a

Source Data Extended Data Fig./Table 2

Statistical Source Data

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Suzuki, K., Goto, Y., Otomo, A. et al. Na+-V-ATPase inhibitor curbs VRE growth and unveils Na+ pathway structure. Nat Struct Mol Biol 32, 450–458 (2025). https://doi.org/10.1038/s41594-024-01419-y

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