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PreprintCited by 1Drug Transport and Resistance MechanismsAntibiotic Resistance in BacteriaBacterial Genetics and Biotechnology

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One-step drug transport across two membranes of Gram-negative bacteria by an MFS tripartite assembly

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Luisi, Ben, Du, Dijun, Chao, Yanjie, Zhong, Zhaojun, Maimaiti, Tuerxunjiang, Jackson, Matthew, Dong, Rui, Gao, Xueyan, Guo, Jinliang, Li, Shangrong, Shang, Wenyu, Liu, Huajun, Jiang, Hongnian, Zhang, Shuo, Zhao, Huimin, Ouyang, Qing·2025·Nature Communications
DOI 10.21203/rs.3.rs-5901545/v1

Preprint — published as 10.1038/s41467-026-70500-5

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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 envelope, often via an indirect stepwise procedure. The major facilitator superfamily (MFS)-type tripartite efflux pumps and type I secretion systems likely employ a direct one-step transport approach, but the 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 unique, asymmetric architecture emerging from the assembly of TolC:EmrA:EmrB with a ratio of 3:6:1. This structure reveals two critical subdomains AssA and AssB that are essential for the pump assembly. Key residues involved in drug recognition, proton translocation and coupling are identified and corroborated by mutagenesis and antibiotic sensitivity assays. The delineation of the complete translocation pathway unravels the one-step drug transport mechanism of the MFS pump across the entire envelope of Gram-negative bacteria.

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Metadata

Authors
Luisi, B., Du, D., Chao, Y., Zhong, Z., Maimaiti, T., Jackson, M., Dong, R., Gao, X., Guo, J., Li, S., Shang, W., Liu, H., Jiang, H., Zhang, S., Zhao, H., & Ouyang, Q.
Affiliations
  • University of Cambridge (GB)
  • Zhengzhou University (CN)
  • Chinese Academy of Sciences (CN)
  • ShanghaiTech University (CN)
  • ShanghaiTech University
Corresponding author
  • Luisi, Ben
  • Du, Dijun
  • Chao, Yanjie
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-02-13
Volume
17
Issue
1
ISSN
2041-1723 (ISSN-L)
DOI
10.21203/rs.3.rs-5901545/v1
OpenAlex
W7136633992
Cited by
1
References
2
Citations by year
2026: 1
FWCI
2.93
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Discovery Research Portal (University of Dundee) (submittedVersion, cc-by)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Apollo (cc-by)
Funders
  • Wellcome Trust (200873/Z/16/Z, 200873)
  • National Natural Science Foundation of China (2022YFC2303200, 92478118, 32270064, 31971133, XDB0570000)
  • Chinese Academy of Sciences (XDB0570000, 176002GJHZ2022022MI)
  • Science and Technology Commission of Shanghai Municipality (24ZR1493200, 22WZ2504100, 19PJ1407900, 19JC1414000)
  • ShanghaiTech University
  • Medical Research Council (RG94521)
  • H2020 European Research Council (742210)
Topics
  • Health Sciences › Medicine › Oncology › Drug Transport and Resistance Mechanisms
  • Health Sciences › Medicine › Microbiology (medical) › Antibiotic Resistance in Bacteria
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
major facilitator superfamily, tripartite efflux pump, TolC, antimicrobial resistance, drug transport mechanisms, crystal structure
MeSH terms
Anti-Bacterial Agents, Bacterial Outer Membrane Proteins, Biological Transport, Cell Membrane, Escherichia coli, Gram-Negative Bacteria, Models, Molecular, Crystallography, X-Ray, Membrane Transport Proteins, Escherichia coli Proteins

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