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Protonation drives the conformational switch in the multidrug transporter LmrP

Nature Chemical Biology volume 10, pages 149–155 (2014)Cite this article

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Abstract

Multidrug antiporters of the major facilitator superfamily couple proton translocation to the extrusion of cytotoxic molecules. The conformational changes that underlie the transport cycle and the structural basis of coupling of these transporters have not been elucidated. Here we used extensive double electron-electron resonance measurements to uncover the conformational equilibrium of LmrP, a multidrug transporter from Lactococcus lactis, and to investigate how protons and ligands shift this equilibrium to enable transport. We find that the transporter switches between outward-open and outward-closed conformations, depending on the protonation states of specific acidic residues forming a transmembrane protonation relay. Our data can be framed in a model of transport wherein substrate binding initiates the transport cycle by opening the extracellular side. Subsequent protonation of membrane-embedded acidic residues induces substrate release to the extracellular side and triggers a cascade of conformational changes that concludes in proton release to the intracellular side.

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Figure 1: The extracellular side of LmrP closes at low pH.
Figure 2: Acidic pH opens the intracellular side of LmrP.
Figure 3: The conformational equilibrium of LmrP is coupled to the protonation of acidic residues.
Figure 4: Hoechst 33342 binding restricts TM8 conformational flexibility and stabilizes the outward-open conformation.
Figure 5: Proposed LmrP transport cycle based on DEER distance measurements.

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References

  1. Li, X.-Z. & Nikaido, H. Efflux-mediated drug resistance in bacteria: an update. Drugs 69, 1555–1623 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Putman, M., van Veen, H.W., Degener, J.E. & Konings, W.N. The lactococcal secondary multidrug transporter LmrP confers resistance to lincosamides, macrolides, streptogramins and tetracyclines. Microbiology 147, 2873–2880 (2001).

    Article  CAS  PubMed  Google Scholar 

  3. Jardetzky, O. Simple allosteric model for membrane pumps. Nature 211, 969–970 (1966).

    Article  CAS  PubMed  Google Scholar 

  4. Tanford, C. Mechanism of free energy coupling in active transport. Annu. Rev. Biochem. 52, 379–409 (1983).

    Article  CAS  PubMed  Google Scholar 

  5. Forrest, L.R., Krämer, R. & Ziegler, C. The structural basis of secondary active transport mechanisms. Biochim. Biophys. Acta 1807, 167–188 (2011).

    Article  CAS  PubMed  Google Scholar 

  6. Reddy, V.S., Shlykov, M.A., Castillo, R., Sun, E.I. & Saier, M.H. The major facilitator superfamily (MFS) revisited. FEBS J. 279, 2022–2035 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Huang, Y., Lemieux, M.J., Song, J., Auer, M. & Wang, D.-N. Structure and mechanism of the glycerol-3-phosphate transporter from Escherichia coli. Science 301, 616–620 (2003).

    Article  CAS  PubMed  Google Scholar 

  8. Guan, L., Mirza, O., Verner, G., Iwata, S. & Kaback, H.R. Structural determination of wild-type lactose permease. Proc. Natl. Acad. Sci. USA 104, 15294–15298 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  9. Dang, S. et al. Structure of a fucose transporter in an outward-open conformation. Nature 467, 734–738 (2010).

    Article  CAS  PubMed  Google Scholar 

  10. Newstead, S. et al. Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J. 30, 417–426 (2011).

    Article  CAS  PubMed  Google Scholar 

  11. Yin, Y., He, X., Szewczyk, P., Nguyen, T. & Chang, G. Structure of the multidrug transporter EmrD from Escherichia coli. Science 312, 741–744 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sun, L. et al. Crystal structure of a bacterial homologue of glucose transporters GLUT1–4. Nature 490, 361–366 (2012).

    Article  CAS  PubMed  Google Scholar 

  13. Lemieux, M.J., Huang, Y. & Wang, D.-N. The structural basis of substrate translocation by the Escherichia coli glycerol-3-phosphate transporter: a member of the major facilitator superfamily. Curr. Opin. Struct. Biol. 14, 405–412 (2004).

    Article  CAS  PubMed  Google Scholar 

  14. Holyoake, J. & Sansom, M.S.P. Conformational change in an MFS protein: MD simulations of LacY. Structure 15, 873–884 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Enkavi, G. & Tajkhorshid, E. Simulation of spontaneous substrate binding revealing the binding pathway and mechanism and initial conformational response of GlpT. Biochemistry 49, 1105–1114 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. Liu, Z., Madej, M.G. & Kaback, H.R. Helix dynamics in LacY: helices II and IV. J. Mol. Biol. 396, 617–626 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhou, Y., Madej, M.G., Guan, L., Nie, Y. & Kaback, H.R. An early event in the transport mechanism of LacY protein: interaction between helices V and I. J. Biol. Chem. 286, 30415–30422 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kaback, H.R., Smirnova, I., Kasho, V., Nie, Y. & Zhou, Y. The alternating access transport mechanism in LacY. J. Membr. Biol. 239, 85–93 (2011).

    Article  CAS  PubMed  Google Scholar 

  19. Smirnova, I., Kasho, V., Sugihara, J., Vázquez-Ibar, J.L. & Kaback, H.R. Role of protons in sugar binding to LacY. Proc. Natl. Acad. Sci. USA 109, 16835–16840 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Yin, Y., Jensen, M.Ø., Tajkhorshid, E. & Schulten, K. Sugar binding and protein conformational changes in lactose permease. Biophys. J. 91, 3972–3985 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Smirnova, I. et al. Sugar binding induces an outward facing conformation of LacY. Proc. Natl. Acad. Sci. USA 104, 16504–16509 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  22. Guan, L. & Kaback, H.R. Binding affinity of lactose permease is not altered by the H+ electrochemical gradient. Proc. Natl. Acad. Sci. USA 101, 12148–12152 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Fluman, N., Ryan, C.M., Whitelegge, J.P. & Bibi, E. Dissection of mechanistic principles of a secondary multidrug efflux protein. Mol. Cell 47, 777–787 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Mazurkiewicz, P., Driessen, A.J.M. & Konings, W.N. Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis. Biochim. Biophys. Acta 1658, 252–261 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Lewinson, O. et al. The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions. Proc. Natl. Acad. Sci. USA 100, 1667–1672 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bolhuis, H. et al. Energetics and mechanism of drug transport mediated by the lactococcal multidrug transporter LmrP. J. Biol. Chem. 271, 24123–24128 (1996).

    Article  CAS  PubMed  Google Scholar 

  27. Jeschke, G. DEER distance measurements on proteins. Annu. Rev. Phys. Chem. 63, 419–446 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. McHaourab, H.S., Steed, P.R. & Kazmier, K. Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy. Structure 19, 1549–1561 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Zou, P. & McHaourab, H.S. Increased sensitivity and extended range of distance measurements in spin-labeled membrane proteins: Q-band double electron-electron resonance and nanoscale bilayers. Biophys. J. 98, L18–L20 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Putman, M., Koole, L.A., van Veen, H.W. & Konings, W.N. The secondary multidrug transporter LmrP contains multiple drug interaction sites. Biochemistry 38, 13900–13905 (1999).

    Article  CAS  PubMed  Google Scholar 

  31. Mazurkiewicz, P., Konings, W.N. & Poelarends, G.J. Acidic residues in the lactococcal multidrug efflux pump LmrP play critical roles in transport of lipophilic cationic compounds. J. Biol. Chem. 277, 26081–26088 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Andersen, A.Z. et al. The metabolic pH response in Lactococcus lactis: an integrative experimental and modelling approach. Comput. Biol. Chem. 33, 71–83 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Hakizimana, P., Masureel, M., Gbaguidi, B., Ruysschaert, J.-M. & Govaerts, C. Interactions between phosphatidylethanolamine headgroup and LmrP, a multidrug transporter: a conserved mechanism for proton gradient sensing? J. Biol. Chem. 283, 9369–9376 (2008).

    Article  CAS  PubMed  Google Scholar 

  34. Seol, W. & Shatkin, A.J. Site-directed mutants of Escherichia coli α-ketoglutarate permease (KgtP). Biochemistry 31, 3550–3554 (1992).

    Article  CAS  PubMed  Google Scholar 

  35. Pazdernik, N.J., Matzke, E.A., Jessen-Marshall, A.E. & Brooker, R.J. Roles of charged residues in the conserved motif, G-X-X-X-D/E-R/K-X-G-[X]-R/K-R/K, of the lactose permease of Escherichia coli. J. Membr. Biol. 174, 31–40 (2000).

    Article  CAS  PubMed  Google Scholar 

  36. Yamaguchi, A., Someya, Y. & Sawai, T. Metal-tetracycline/H+ antiporter of Escherichia coli encoded by transposon Tn10. The role of a conserved sequence motif, GXXXXRXGRR, in a putative cytoplasmic loop between helices 2 and 3. J. Biol. Chem. 267, 19155–19162 (1992).

    CAS  PubMed  Google Scholar 

  37. Schaedler, T.A. & van Veen, H.W. A flexible cation binding site in the multidrug major facilitator superfamily transporter LmrP is associated with variable proton coupling. FASEB J. 24, 3653–3661 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Gbaguidi, B. et al. Proton motive force mediates a reorientation of the cytosolic domains of the multidrug transporter LmrP. Cell Mol. Life Sci. 61, 2646–2657 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Gbaguidi, B., Hakizimana, P., Vandenbussche, G. & Ruysschaert, J.-M. Conformational changes in a bacterial multidrug transporter are phosphatidylethanolamine-dependent. Cell Mol. Life Sci. 64, 1571–1582 (2007).

    Article  CAS  PubMed  Google Scholar 

  40. Mazurkiewicz, P., Poelarends, G.J., Driessen, A.J.M. & Konings, W.N. Facilitated drug influx by an energy-uncoupled secondary multidrug transporter. J. Biol. Chem. 279, 103–108 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Madej, M.G., Soro, S.N. & Kaback, H.R. Apo-intermediate in the transport cycle of lactose permease (LacY). Proc. Natl. Acad. Sci. USA 109, E2970–E2978 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Hirai, T. & Subramaniam, S. Structure and transport mechanism of the bacterial oxalate transporter OxlT. Biophys. J. 87, 3600–3607 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Bolhuis, H. et al. Multidrug resistance in Lactococcus lactis: evidence for ATP-dependent drug extrusion from the inner leaflet of the cytoplasmic membrane. EMBO J. 15, 4239–4245 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Shapiro, A.B. & Ling, V. Extraction of Hoechst 33342 from the cytoplasmic leaflet of the plasma membrane by P-glycoprotein. Eur. J. Biochem. 250, 122–129 (1997).

    Article  CAS  PubMed  Google Scholar 

  45. Mitchell, B.A., Paulsen, I.T., Brown, M.H. & Skurray, R.A. Bioenergetics of the staphylococcal multidrug export protein QacA. Identification of distinct binding sites for monovalent and divalent cations. J. Biol. Chem. 274, 3541–3548 (1999).

    Article  CAS  PubMed  Google Scholar 

  46. Ocaktan, A., Yoneyama, H. & Nakae, T. Use of fluorescence probes to monitor function of the subunit proteins of the MexA-MexB-oprM drug extrusion machinery in Pseudomonas aeruginosa. J. Biol. Chem. 272, 21964–21969 (1997).

    Article  CAS  PubMed  Google Scholar 

  47. Baker, J., Wright, S.H. & Tama, F. Simulations of substrate transport in the multidrug transporter EmrD. Proteins 80, 1620–1632 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Frillingos, S. & Kaback, H.R. The role of helix VIII in the lactose permease of Escherichia coli: II. Site-directed sulfhydryl modification. Protein Sci. 6, 438–443 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Soskine, M., Adam, Y. & Schuldiner, S. Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter. J. Biol. Chem. 279, 9951–9955 (2004).

    Article  CAS  PubMed  Google Scholar 

  50. Lu, M. et al. Structures of a Na+-coupled, substrate-bound MATE multidrug transporter. Proc. Natl. Acad. Sci. USA 110, 2099–2104 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Thompson, J.D., Higgins, D.G. & Gibson, T.J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Eswar, N. et al. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. Chapter 2 Unit 2.9 (2007).

  53. Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Putman, M., van Veen, H.W., Poolman, B. & Konings, W.N. Restrictive use of detergents in the functional reconstitution of the secondary multidrug transporter LmrP. Biochemistry 38, 1002–1008 (1999).

    Article  CAS  PubMed  Google Scholar 

  55. Jeschke, G. Distance measurements in the nanometer range by pulse EPR. ChemPhysChem 3, 927–932 (2002).

    Article  CAS  PubMed  Google Scholar 

  56. Jeschke, G. et al. DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data. Appl. Magn. Reson. 30, 473–498 (2006).

    Article  CAS  Google Scholar 

  57. Chiang, Y.-W., Borbat, P.P. & Freed, J.H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–295 (2005).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank H. Remaut for insightful discussions and H. Koteiche and R. Steed for critically reading the manuscript. This work was supported by a Mandat d'Impulsion Scientifique (no. F.4523.12) from the Fonds de la Recherche Scientifique (FRS-FNRS), Belgium, to C.G. and by the National Institute for General Medical Sciences–US National Institutes of Health (GM-077659) to H.S.M. M.M. was a research fellow of the Fonds pour la Formation à la Recherche dans l′Industrie et dans l′Agriculture (FRIA), Belgium, and the FRS-FNRS. C.M. is a research fellow of the FRIA. C.G. is a research associate of the FRS-FNRS.

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Author notes

  1. Matthieu Masureel and Chloé Martens: These authors contributed equally to this work.

  2. Hassane S Mchaourab and Cédric Govaerts: These authors are co-last authors.

Authors and Affiliations

  1. Laboratory for the Structure and Function of Biological Membranes, Center for Structural Biology and Bioinformatics, Université Libre de Bruxelles, Brussels, Belgium

    Matthieu Masureel, Chloé Martens, Jean-Marie Ruysschaert & Cédric Govaerts

  2. Department of Molecular Physiology and Biophysics, Vanderbilt University Medical Center, Nashville, Tennessee, USA

    Richard A Stein, Smriti Mishra & Hassane S Mchaourab

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  1. Matthieu Masureel
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Contributions

M.M., C.M., H.S.M. and C.G. were involved in experimental design. M.M. and C.M. performed mutagenesis, expression, activity, purification and labeling experiments. M.M., R.A.S., S.M. and H.S.M. performed EPR measurements. R.A.S. and H.M. performed DEER and continuous-wave data analysis. C.G. and C.M. performed molecular modeling. C.G. and H.M. oversaw all aspects of the experiments and manuscript preparation. All authors participated in interpreting the data and writing the paper.

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Correspondence to Hassane S Mchaourab or Cédric Govaerts.

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Masureel, M., Martens, C., Stein, R. et al. Protonation drives the conformational switch in the multidrug transporter LmrP. Nat Chem Biol 10, 149–155 (2014). https://doi.org/10.1038/nchembio.1408

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