Abstract
Transport of proteins and small molecules across cellular membrane is crucial for bacterial interaction with the environment and survival against antibiotics. In Gram-negative bacteria that possess two layers of membranes, specialized macromolecular machines are required to transport substrates across the cell envelope, often via an indirect stepwise process. The major facilitator superfamily (MFS)-type tripartite efflux pumps use proton electrochemical gradient to extrude drugs in diverse bacterial species, but the architecture of the assembly and structural mechanisms remain elusive. A representative MFS-type tripartite efflux pump, EmrAB-TolC, mediates resistance to multiple antimicrobial drugs through proton-coupled EmrB, a member of the DHA2 transporter family. Here, we report the high-resolution (3.13 Å) structure of the EmrAB-TolC pump, revealing a distinct, asymmetric architecture emerging from the assembly of TolC:EmrA:EmrB with a ratio of 3:6:1 and contacts that are essential for the pump assembly. Key residues involved in drug transport are identified and corroborated by mutagenesis and antibiotic sensitivity assays. The structural and functional data support a model for one-step drug transport by the MFS pump across the entire envelope of Gram-negative bacteria.
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Data availability
Coordinates have been deposited in the Protein Data Bank (PDB) under PDB codes 8ZAL (EmrAB-TolC pump-EA) and 8ZAR (EmrAB-TolC pump-FA). Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMD) under EMD codes EMD-39879 (EmrAB-TolC pump-EA) and EMD-39885 (EmrAB-TolC pump-FA). Molecular dynamics data are in the Figshare repository with the doi: 10.6084/m9.figshare.31240081 [doi.org/10.6084/m9.figshare.31240081]. Source data are provided with this paper.
Code availability
Molecular Dynamics input files, coordinates and simulation trajectories can be accessed at doi: 10.6084/m9.figshare.31240081 [doi.org/10.6084/m9.figshare.31240081].
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Acknowledgements
This work was supported by the National Key R&D Program of China (2022YFC2303200); National Natural Science Foundation of China (31971133 to D.D.; 32270064 and 92478118 to Y.C.), the Science and Technology Commission of Shanghai Municipality (19PJ1407900, 19JC1414000 and 22WZ2504100 to D.D.; 24ZR1493200 to Y.C.), and the Chinese Academy of Sciences (XDB0570000 and 176002GJHZ2022022MI to Y.C.). BFL was supported by ERC Advanced grant (742210) and a Wellcome Trust Investigator award (200873/Z/16/Z). MLJ is supported by a UK Medical Research Council Intramural Programme Award MC_UU_000254/4 (RG94521).. Cryo-EM data were collected at the Bio-Electron Microscopy Facility of ShanghaiTech University with the assistance of Q. Sun, D. Liu, Z. Zhang, L. Wang and Y. Yang. We thank the Molecular Imaging Core Facility, the Molecular and Cell Biology Core Facility, and the Multi-Omics Core Facility at the School of Life Science and Technology for providing technical support. We are also grateful for the support of Lajos Kalmar of the MRC Toxicology Unit in the use of high-performance computing used in this study. We thank Sofiya Mason for help with molecular docking.
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Z.Z. performed cloning and overexpression of the EmrAB-TolC complex; Z.Z. and T.M. purified the EmrAB-TolC complex, prepared cryo-EM samples, collected cryo-EM data, determined structures, performed model building and refinement and prepared figures for the manuscript; J.G. and X.G. carried out drug-proton antiport assay; W.W., X.G., W.S., Q.W., J.G., S.L., H.L. and Q.O. optimized in-column peptide-disc methods, prepared homemade graphene monolayer grids, and assisted the collection of cryo-EM data; R.D., H.J., Z.Z., T.M., X.G., S.Z. and W.S. performed antibiotics sensitivity assays; T.M. and H.L. carried out Western blot assay; Y.C. supervised antibiotics sensitivity assays and discussed project design; M.L.J. performed protein structure predictions and modeling; U.Z. performed and analysed the molecular dynamics simulations; D.D. and B.L. conceived the project; D.D. designed and supervised all experiments; D.D., Y.C. and B.L. wrote the manuscript. All the authors contributed to the data interpretation and manuscript preparation.
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Zhong, Z., Maimaiti, T., Jackson, M.L. et al. A model for drug transport across two membranes of Gram-negative bacteria by an MFS tripartite assembly. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70500-5
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DOI: https://doi.org/10.1038/s41467-026-70500-5
