Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport

Abstract

Gram-negative bacteria, such as Escherichia coli, frequently use tripartite efflux complexes in the resistance-nodulation-cell division (RND) family to expel various toxic compounds from the cell1,2. The efflux system CusCBA is responsible for extruding biocidal Cu(I) and Ag(I) ions3,4. No previous structural information was available for the heavy-metal efflux (HME) subfamily of the RND efflux pumps. Here we describe the crystal structures of the inner-membrane transporter CusA in the absence and presence of bound Cu(I) or Ag(I). These CusA structures provide new structural information about the HME subfamily of RND efflux pumps. The structures suggest that the metal-binding sites, formed by a three-methionine cluster, are located within the cleft region of the periplasmic domain. This cleft is closed in the apo-CusA form but open in the CusA-Cu(I) and CusA-Ag(I) structures, which directly suggests a plausible pathway for ion export. Binding of Cu(I) and Ag(I) triggers significant conformational changes in both the periplasmic and transmembrane domains. The crystal structure indicates that CusA has, in addition to the three-methionine metal-binding site, four methionine pairs—three located in the transmembrane region and one in the periplasmic domain. Genetic analysis and transport assays suggest that CusA is capable of actively picking up metal ions from the cytosol, using these methionine pairs or clusters to bind and export metal ions. These structures suggest a stepwise shuttle mechanism for transport between these sites.

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: Structure of the apo CusA efflux pump.
Figure 2: Comparison of the apo and metal-bound structures of CusA.
Figure 3: Anomalous maps of the bound metal ions.
Figure 4: Proposed metal transport pathway of the CusA efflux pump.
Figure 5: Stopped-flow transport assay of reconstituted CusA with extravesicular Ag + ion.

References

  1. 1

    Tseng, T. T. et al. The RND permease superfamily: an ancient, ubiquitous and diverse family that includes human disease and development protein. J. Mol. Microbiol. Biotechnol. 1, 107–125 (1999)

    CAS  PubMed  Google Scholar 

  2. 2

    Nies, D. H. Efflux-mediated heavy metal resistance in prokaryotes. FEMS Microbiol. Rev. 27, 313–339 (2003)

    CAS  Article  Google Scholar 

  3. 3

    Franke, S., Grass, G. & Nies, D. H. The product of the ybdE gene of the Escherichia coli chromosome is involved in detoxification of silver ions. Microbiology 147, 965–972 (2001)

    CAS  Article  Google Scholar 

  4. 4

    Franke, S., Grass, G., Rensing, C. & Nies, D. H. Molecular analysis of the copper-transporting efflux system CusCFBA of Escherichia coli . J. Bacteriol. 185, 3804–3812 (2003)

    Article  Google Scholar 

  5. 5

    Murakami, S., Nakashima, R., Yamashita, E. & Yamaguchi, A. Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593 (2002)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Yu, E. W., McDermott, G., Zgruskaya, H. I., Nikaido, H. & Koshland, D. E., Jr Structural basis of multiple drug binding capacity of the AcrB multidrug efflux pump. Science 300, 976–980 (2003)

    ADS  CAS  Article  Google Scholar 

  7. 7

    Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T. & Yamaguchi, A. Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179 (2006)

    ADS  CAS  Article  Google Scholar 

  8. 8

    Seeger, M. A. et al. Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298 (2006)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Sennhauser, G., Amstutz, P., Briand, C., Storchengegger, O. & Grütter, M. G. Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol. 5, e7 (2007)

    Article  Google Scholar 

  10. 10

    Yu, E. W., Aires, J. R., McDermott, G. & Nikaido, H. A periplasmic-drug binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study. J. Bacteriol. 187, 6804–6815 (2005)

    CAS  Article  Google Scholar 

  11. 11

    Su, C.-C. et al. Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway. J. Bacteriol. 188, 7290–7296 (2006)

    CAS  Article  Google Scholar 

  12. 12

    Sennhauser, G., Bukowska, M. A., Briand, C. & Grütter, M. G. Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa . J. Mol. Biol. 389, 134–145 (2009)

    CAS  Article  Google Scholar 

  13. 13

    Koronakis, V., Sharff, A., Koronakis, E., Luisi, B. & Hughes, C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature 405, 914–919 (2000)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Akama, H. et al. Crystal structure of the drug discharge outer membrane protein, OprM, of Pseudomonas aeruginosa . J. Biol. Chem. 279, 52816–52819 (2004)

    CAS  Article  Google Scholar 

  15. 15

    Mikolosko, J., Bobyk, K., Zgurskaya, H. I. & Ghosh, P. Conformational flexibility in the multidrug efflux system protein AcrA. Structure 14, 577–587 (2006)

    CAS  Article  Google Scholar 

  16. 16

    Higgins, M. K., Bokma, E., Koronakis, E., Hughes, C. & Koronakis, V. Structure of the periplasmic component of a bacterial drug efflux pump. Proc. Natl Acad. Sci. USA 101, 9994–9999 (2004)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Akama, H. et al. Crystal structure of the membrane fusion protein, MexA, of the multidrug transporter in Pseudomonas aeruginosa . J. Biol. Chem. 279, 25939–25942 (2004)

    CAS  Article  Google Scholar 

  18. 18

    Symmons, M., Bokma, E., Koronakis, E., Hughes, C. & Koronakis, V. The assembled structure of a complete tripartite bacterial multidrug efflux pump. Proc. Natl Acad. Sci. USA 106, 7173–7178 (2009)

    ADS  CAS  Article  Google Scholar 

  19. 19

    Su, C.-C. et al. Crystal structure of the membrane fusion protein CusB from Escherichia coli . J. Mol. Biol. 393, 342–355 (2009)

    CAS  Article  Google Scholar 

  20. 20

    Zhou, H. & Thiele, D. J. Identification of a novel high affinity copper transport complex in the fission yeast Schizosaccharomyces pombe . J. Biol. Chem. 276, 20529–20535 (2001)

    CAS  Article  Google Scholar 

  21. 21

    Jiang, J., Nadas, I. A., Kim, M. A. & Franz, K. J. A Mets motif peptide found in copper transport proteins selectively binds Cu(I) with methionine-only coordination. Inorg. Chem. 44, 9787–9794 (2005)

    CAS  Article  Google Scholar 

  22. 22

    Banci, L. et al. The Atx1–Ccc2 complex is a metal-mediated protein–protein interaction. Nature Chem. Biol. 2, 367–368 (2006)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Xue, Y. et al. Cu(I) recognition via cation-π and methionine interactions in CusF. Nature Chem. Biol. 4, 107–109 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Loftin, I. R., Franke, S., Blackburn, N. J. & McEvoy, M. M. Unusual Cu(I)/Ag(I) coordination of Escherichia coli CusF as revealed by atomic resolution crystallography and x-ray absorption spectroscopy. Protein Sci. 16, 2287–2293 (2007)

    CAS  Article  Google Scholar 

  25. 25

    Changela, A. et al. Molecular basis of metal-ion selectivity and zeptomolar sensitivity by CueR. Science 301, 1383–1387 (2003)

    ADS  CAS  Article  Google Scholar 

  26. 26

    Arnesano, F., Banci, L., Bertini, I., Huffman, D. L. & O’Halloran, T. V. Solution structure of the Cu(I) and apo-forms of the yeast metallochaperone, Atx1. Biochemistry 40, 1528–1539 (2001)

    CAS  Article  Google Scholar 

  27. 27

    Atilgan, A. R. et al. Anisotropy of fluctuation dynamics of proteins with an elastic network model. Biophys. J. 80, 505–515 (2001)

    ADS  CAS  Article  Google Scholar 

  28. 28

    Goldberg, M., Pribyl, T., Juhuke, S. & Nies, D. H. Energetics and topology of CzcA, a cation/proton antiporter of the resistance-nodulation-cell division protein family. J. Biol. Chem. 274, 26065–26070 (1999)

    CAS  Article  Google Scholar 

  29. 29

    Aires, J. R. & Nikaido, H. Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli . J. Bacteriol. 187, 1923–1929 (2005)

    CAS  Article  Google Scholar 

  30. 30

    Otwinowski, Z. & Minor, M. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  Article  Google Scholar 

  31. 31

    Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    Article  Google Scholar 

  32. 32

    Pape, T. & Schneider, T. R. HKL2MAP: a graphical user interface for macromolecular phasing with SHELX programs. J. Appl. Cryst. 37, 843–844 (2004)

    CAS  Article  Google Scholar 

  33. 33

    Otwinowski, Z. MLPHARE, CCP4 Proc. 80 (Daresbury Laboratory, 1991)

    Google Scholar 

  34. 34

    Collaborative Computational Project No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

  35. 35

    Terwilliger, T. C. Maximum-likelihood density modification using pattern recognition of structural motifs. Acta Crystallogr. D 57, 1755–1762 (2001)

    CAS  Article  Google Scholar 

  36. 36

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  37. 37

    Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. 58, 1948–1954 (2002)

    Google Scholar 

  38. 38

    Brünger, A. T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  Google Scholar 

  39. 39

    McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007)

    CAS  Article  Google Scholar 

  40. 40

    Gabb, H. A., Jackson, R. M. & Sternberg, M. J. E. Modelling protein docking using shape complementarity, electrostatics, and biochemical information. J. Mol. Biol. 272, 106–120 (1997)

    CAS  Article  Google Scholar 

  41. 41

    Katchalski-Katzir, E. et al. Molecular surface recognition: determination of geometric fit between proteins and their ligands by correlation techniques. Proc. Natl Acad. Sci. USA 89, 2195–2199 (1992)

    ADS  CAS  Article  Google Scholar 

  42. 42

    Phillips, J. C. et al. Scalable molecular dynamics with NAMD. J. Comput. Chem. 26, 1781–1802 (2005)

    CAS  Article  Google Scholar 

  43. 43

    Feller, S. E. & MacKerell, A. D., Jr An improved empirical potential energy for molecular simulations of phospholipids. J. Phys. Chem. B 104, 7510–7515 (2000)

    CAS  Article  Google Scholar 

  44. 44

    Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl Acad. Sci. USA 97, 6640–6645 (2000)

    ADS  CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank M. D. Routh for critical reading of the manuscript. This work is based on research conducted at the Northeastern Collaborative Access Team beamlines of the Advanced Photon Source, supported by National Institutes of Health (NIH) award RR-15301 from the National Center for Research Resources. Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Basic Energy Sciences, under contract no. DE-AC02-06CH11357. This work was supported by NIH grants GM 074027 (to E.W.Y.), GM 086431 (to E.W.Y.), GM 081680 (to R.L.J.) and GM 072014 (to R.L.J.).

Author information

Affiliations

Authors

Contributions

F.L., C.-C.S. and E.W.Y. designed the research. F.L. and C.-C.S. performed experiments. M.T.Z. and S.E.B. performed simulations. C.-C.S., F.L., K.R.R. and E.W.Y. performed model building and refinement. F.L., C.-C.S., R.L.J. and E.W.Y. wrote the paper.

Corresponding author

Correspondence to Edward W. Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Figures 1-14 plus legends and Supplementary Tables 1-4. (PDF 17188 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Long, F., Su, C., Zimmermann, M. et al. Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature 467, 484–488 (2010). https://doi.org/10.1038/nature09395

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.