Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump

Abstract

The MacA–MacB–TolC assembly of Escherichia coli is a transmembrane machine that spans the cell envelope and actively extrudes substrates, including macrolide antibiotics and polypeptide virulence factors. These transport processes are energized by the ATPase MacB, a member of the ATP-binding cassette (ABC) superfamily. We present an electron cryo-microscopy structure of the ABC-type tripartite assembly at near-atomic resolution. A hexamer of the periplasmic protein MacA bridges between a TolC trimer in the outer membrane and a MacB dimer in the inner membrane, generating a quaternary structure with a central channel for substrate translocation. A gating ring found in MacA is proposed to act as a one-way valve in substrate transport. The MacB structure features an atypical transmembrane domain with a closely packed dimer interface and a periplasmic opening that is the likely portal for substrate entry from the periplasm, with subsequent displacement through an allosteric transport mechanism.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: A pseudo-atomic model for the MacAB–TolC pump.
Figure 2: Structure of MacB.
Figure 3: Interactions between MacA and MacB.

References

  1. 1

    Kobayashi, N., Nishino, K. & Yamaguchi, A. Novel macrolide-specific ABC-type efflux transporter in Escherichia coli. J. Bacteriol. 183, 5639–5644 (2001).

    CAS  Article  Google Scholar 

  2. 2

    Vallet-Gely, I. et al. Association of hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, with cyclic lipopeptide production. Appl. Environ. Microbiol. 76, 910–921 (2010).

    CAS  Article  Google Scholar 

  3. 3

    Cho, H. & Kang, H. The PseEF efflux system is a virulence factor of Pseudomonas syringae pv. syringae. J. Microbiol. 50, 79–90 (2012).

    CAS  Article  Google Scholar 

  4. 4

    Turlin, E. et al. Protoporphyrin (PPIX) efflux by the MacAB–TolC pump in Escherichia coli. Microbiologyopen 3, 849–859 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Yamanaka, H., Kobayashi, H., Takahashi, E. & Okamoto, K. MacAB is involved in the secretion of Escherichia coli heat-stable enterotoxin II. J. Bacteriol. 190, 7693–7698 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Lu, S. & Zgurskaya, H. I. MacA, a periplasmic membrane fusion protein of the macrolide transporter MacAB–TolC, binds lipopolysaccharide core specifically and with high affinity. J. Bacteriol. 195, 4865–4872 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Lu, S. & Zgurskaya, H. I. Role of ATP binding and hydrolysis in assembly of MacAB–TolC macrolide transporter. Mol. Microbiol. 86, 1132–1143 (2012).

    CAS  Article  Google Scholar 

  8. 8

    Tikhonova, E. B., Devroy, V. K., Lau, S. Y. & Zgurskaya, H. I. Reconstitution of the Escherichia coli macrolide transporter: the periplasmic membrane fusion protein MacA stimulates the ATPase activity of MacB. Mol. Microbiol. 63, 895–910 (2007).

    CAS  Article  Google Scholar 

  9. 9

    Dawson, R. J. P. & Locher, K. P. Structure of a bacterial multidrug ABC transporter. Nature 443, 180–185 (2006).

    CAS  Article  Google Scholar 

  10. 10

    Ward, A., Reyes, C. L., Yu, J., Roth, C. B. & Chang, G. Flexibility in the ABC transporter MsbA: alternating access with a twist. Proc. Natl Acad. Sci. USA 104, 19005–19010 (2007).

    CAS  Article  Google Scholar 

  11. 11

    Gutmann, D. A. P., Ward, A., Urbatsch, I. L., Chang, G. & van Veen, H. W. Understanding polyspecificity of multidrug ABC transporters: closing in on the gaps in ABCB1. Trends Biochem. Sci. 35, 36–42 (2010).

    CAS  Article  Google Scholar 

  12. 12

    Doshi, R., Woebking, B. & van Veen, H. W. Dissection of the conformational cycle of the multidrug/lipidA ABC exporter MsbA. Proteins 78, 2867–2872 (2010).

    CAS  Article  Google Scholar 

  13. 13

    Choudhury, H. G. et al. Structure of an antibacterial peptide ATP-binding cassette transporter in a novel outward occluded state. Proc. Natl Acad. Sci. USA 111, 9145–9150 (2014).

    CAS  Article  Google Scholar 

  14. 14

    Lee, J.-Y. et al. Crystal structure of the human sterol transporter ABCG5/ABCG8. Nature 533, 561–564 (2016).

    CAS  Article  Google Scholar 

  15. 15

    Aittoniemi, J. et al. SUR1: a unique ATP-binding cassette protein that functions as an ion channel regulator. Philos. Trans. R. Soc. B 364, 257–267 (2009).

    CAS  Article  Google Scholar 

  16. 16

    Locher, K. P. Mechanistic diversity in ATP-binding cassette (ABC) transporters. Nat. Struct. Mol. Biol. 23, 487–493 (2016).

    CAS  Article  Google Scholar 

  17. 17

    Perez, C. et al. Structure and mechanism of an active lipid-linked oligosaccharide flippase. Nature 524, 433–438 (2015).

    CAS  Article  Google Scholar 

  18. 18

    Khare, D., Oldham, M. L., Orelle, C., Davidson, A. L. & Chen, J. Alternating access in maltose transporter mediated by rigid-body rotations. Mol. Cell 33, 528–536 (2009).

    CAS  Article  Google Scholar 

  19. 19

    Woo, J.-S., Zeltina, A., Goetz, B. A. & Locher, K. P. X-ray structure of the Yersinia pestis heme transporter HmuUV. Nat. Struct. Mol. Biol. 19, 1310–1315 (2012).

    CAS  Article  Google Scholar 

  20. 20

    Korkhov, V. M., Mireku, S. A. & Locher, K. P. Structure of AMP-PNP-bound vitamin B12 transporter BtuCD-F. Nature 490, 367–372 (2012).

    CAS  Article  Google Scholar 

  21. 21

    Song, S., Kim, J.-S., Lee, K. & Ha, N.-C. Molecular architecture of the bacterial tripartite multidrug efflux pump focusing on the adaptor bridging model. J. Microbiol. 53, 355–364 (2015).

    CAS  Article  Google Scholar 

  22. 22

    Zgurskaya, H. I., Weeks, J. W., Ntreh, A. T., Nickels, L. M. & Wolloscheck, D. Mechanism of coupling drug transport reactions located in two different membranes. Front. Microbiol. 6, 100 (2015).

    PubMed  PubMed Central  Google Scholar 

  23. 23

    Du, D., van Veen, H. W. & Luisi, B. F. Assembly and operation of bacterial tripartite multidrug efflux pumps. Trends Microbiol. 23, 311–319 (2015).

    CAS  Article  Google Scholar 

  24. 24

    Yamanaka, H., Izawa, H. & Okamoto, K. Carboxy-terminal region involved in activity of Escherichia coli TolC. J. Bacteriol. 183, 6961–6964 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Du, D. et al. Structure of the AcrAB–TolC multidrug efflux pump. Nature 509, 512–515 (2014).

    CAS  Article  Google Scholar 

  26. 26

    Daury, L. et al. Tripartite assembly of RND multidrug efflux pumps. Nat. Commun. 7, 10731 (2016).

    CAS  Article  Google Scholar 

  27. 27

    Kim, J.-S. et al. Structure of the tripartite multidrug efflux pump AcrAB–TolC suggests an alternative assembly mode. Mol. Cells 38, 180–186 (2015).

    Article  Google Scholar 

  28. 28

    Lin, H. T. et al. Macb ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA. J. Biol. Chem. 284, 1145–1154 (2009).

    CAS  Article  Google Scholar 

  29. 29

    Xu, Y. et al. Crystal structure of the periplasmic region of MacB, a noncanonic ABC transporter. Biochemistry 48, 5218–5225 (2009).

    CAS  Article  Google Scholar 

  30. 30

    Yum, S. et al. Crystal structure of the periplasmic component of a tripartite macrolide-specific efflux pump. J. Mol. Biol. 387, 1286–1297 (2009).

    CAS  Article  Google Scholar 

  31. 31

    Calladine, C. R., Sharff, A. & Luisi, B. How to untwist an α-helix: structural principles of an α-helical barrel. J. Mol. Biol. 305, 603–618 (2001).

    CAS  Article  Google Scholar 

  32. 32

    Modali, S. D. & Zgurskaya, H. I. The periplasmic membrane proximal domain of MacA acts as a switch in stimulation of ATP hydrolysis by MacB transporter. Mol. Microbiol. 81, 937–951 (2011).

    CAS  Article  Google Scholar 

  33. 33

    Bai, X., Rajendra, E., Yang, G., Shi, Y. & Scheres, S. H. W. Sampling the conformational space of the catalytic subunit of human γ-secretase. eLife 4, e11182 (2015).

    Article  Google Scholar 

  34. 34

    Jeong, H. et al. Pseudoatomic structure of the tripartite multidrug efflux pump AcrAB–TolC reveals the intermeshing cogwheel-like interaction between AcrA and TolC. Structure 24, 272–276 (2016).

    CAS  Article  Google Scholar 

  35. 35

    Xu, Y. et al. The tip region of the MacA α-hairpin is important for the binding to TolC to the Escherichia coli MacAB–TolC pump. Biochem. Biophys. Res. Commun. 394, 962–965 (2010).

    CAS  Article  Google Scholar 

  36. 36

    Xu, Y. et al. Functional implications of an intermeshing cogwheel-like interaction between TolC and MacA in the action of macrolide-specific efflux pump MacAB–TolC. J. Biol. Chem. 286, 13541–13549 (2011).

    CAS  Article  Google Scholar 

  37. 37

    Lee, M. et al. The α-barrel tip region of Escherichia coli TolC homologs of Vibrio vulnificus interacts with the MacA protein to form the functional macrolide-specific efflux pump MacAB–TolC. J. Microbiol. 51, 154–159 (2013).

    CAS  Article  Google Scholar 

  38. 38

    Dong, C. et al. Wza the translocon for E. coli capsular polysaccharides defines a new class of membrane protein. Nature 444, 226–229 (2006).

    CAS  Article  Google Scholar 

  39. 39

    Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).

    CAS  Article  Google Scholar 

  40. 40

    Meyerson, J. R. et al. Self-assembled monolayers improve protein distribution on holey carbon cryo-EM supports. Sci. Rep. 4, 7084 (2014).

  41. 41

    Russo, C. J. & Passmore, L. A. Electron microscopy: ultrastable gold substrates for electron cryomicroscopy. Science 346, 1377–1380 (2014).

    CAS  Article  Google Scholar 

  42. 42

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

    CAS  Article  Google Scholar 

  43. 43

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    CAS  Article  Google Scholar 

  44. 44

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  45. 45

    Scheres, S. H. W. Semi-automated selection of cryo-EM particles in RELION-1.3. J. Struct. Biol. 189, 114–122 (2015).

    CAS  Article  Google Scholar 

  46. 46

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

    CAS  Article  Google Scholar 

  47. 47

    Scheres, S. H. Beam-induced motion correction for sub-megadalton cryo-EM particles. eLife 3, e03665 (2014).

    Article  Google Scholar 

  48. 48

    Scheres, S. H. W. Processing of structurally heterogeneous cryo-EM data in RELION. Methods Enzymol. 579, 125–157 (2016).

    CAS  Article  Google Scholar 

  49. 49

    Scheres, S. H. W. & Chen, S. Prevention of overfitting in cryo-EM structure determination. Nat. Methods 9, 853–854 (2012).

    CAS  Article  Google Scholar 

  50. 50

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

    CAS  Article  Google Scholar 

  51. 51

    Abraham, M. J. et al. GROMACS: high performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX 1, 19–25 (2015).

    Article  Google Scholar 

  52. 52

    Hornak, V. et al. Comparison of multiple AMBER force fields and development of improved protein backbone parameters. Proteins 65, 712–725 (2006).

    CAS  Article  Google Scholar 

  53. 53

    Wang, J., Cieplak, P. & Kollman, P. A. How well does a restrained electrostatic potential (RESP) model perform in calculating conformational energies of organic and biological molecules? J. Comput. Chem. 21, 1049–1074 (2000).

    CAS  Article  Google Scholar 

  54. 54

    Case, D. A. et al. AMBER 12 (Univ. California, 2012).

    Google Scholar 

  55. 55

    Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W. & Klein, M. L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926 (1983).

    CAS  Article  Google Scholar 

  56. 56

    Joung, I. S. & Cheatham, T. E. Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J. Phys. Chem. B 112, 9020–9041 (2008).

    CAS  Article  Google Scholar 

  57. 57

    Grubmüller, H., Heymann, B. & Tavan, P. Ligand binding: molecular mechanics calculation of the streptavidin–biotin rupture force. Science 271, 997–999 (1996).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported by the Wellcome Trust (to B.F.L.), the Human Frontiers Science Program (to B.F.L., H.W.v.V. and S.M.), a Marie Curie International Outgoing Fellowship (to A.W.P.F.), the UK Medical Research Council (MC_UP_A025_1013, to S.H.W.S.), a Wellcome Trust ISSF award (grant no. WT097818MF), the Scottish Universities’ Physics Alliance (to U.Z. and S.L.) and the MRC Mitochondrial Biology Unit (grant no. U105663141). A.N. is the recipient of a Herchel–Smith Scholarship. The authors thank J. Skepper for help using the EM microscope and the imaging facility at Cambridge University, H. Zhou for providing access to the electron microscope at the University of California at Los Angeles, J. Grimmett and T. Darling for support with high-performance computing at the MRC Laboratory of Molecular Biology, S. Rankin for help with computing on the High-Performance Computing System at the University of Cambridge, and L. Packman for mass spectrometry analyses. The authors thank the staff of the Diamond Light Source for access to the eBIC facility. The EM maps have been deposited in the Electron Microscopy Databank.

Author information

Affiliations

Authors

Contributions

D.D., B.F.L. and S.H.W.S. designed the project. D.D. purified the fusion and disulfide-linkage-stabilized MacAB–TolC complexes. D.D., A.W.P.F., X.C.B. and J.N.B. obtained and analysed the single-particle cryo-EM data. U.O. and S.M. built the homology model of MacB. D.D. and B.F.L. devised a model of MacAB–TolC based on the cryo-EM map. A.N. and H.W.v.V. conducted MIC assays on the MacAB–TolC pump. S.L. and U.Z. carried out molecular dynamics simulations of MacA. D.D., B.F.L. and S.H.W.S. wrote the paper. All authors contributed to editing the manuscript.

Corresponding authors

Correspondence to Sjors H. W. Scheres or Ben F. Luisi or Dijun Du.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–14. (PDF 5660 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fitzpatrick, A., Llabrés, S., Neuberger, A. et al. Structure of the MacAB–TolC ABC-type tripartite multidrug efflux pump. Nat Microbiol 2, 17070 (2017). https://doi.org/10.1038/nmicrobiol.2017.70

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing