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Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE

Nature Chemical Biology volume 12, pages 141–145 (2016)Cite this article

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Abstract

Secondary active transport proteins play a central role in conferring bacterial multidrug resistance. In this work, we investigated the proton-coupled transport mechanism for the Escherichia coli drug efflux pump EmrE using NMR spectroscopy. Our results show that the global conformational motions necessary for transport are modulated in an allosteric fashion by the protonation state of a membrane-embedded glutamate residue. These observations directly correlate with the resistance phenotype for wild-type EmrE and the E14D mutant as a function of pH. Furthermore, our results support a model in which the pH gradient across the inner membrane of E. coli may be used on a mechanistic level to shift the equilibrium of the transporter in favor of an inward-open resting conformation poised for drug binding.

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Figure 1: pH-induced conformational changes to EmrE.
Figure 2: Glu14 protonation sets the resting state.
Figure 3: Allosteric conformational change from Glu14 protonation.
Figure 4: Conformational bias from loop 2 mutant in mixed EmrE dimers.

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References

  1. Higgins, C.F. Multiple molecular mechanisms for multidrug resistance transporters. Nature 446, 749–757 (2007).

    Article  CAS  Google Scholar 

  2. Bay, D.C., Rommens, K.L. & Turner, R.J. Small multidrug resistance proteins: a multidrug transporter family that continues to grow. Biochim. Biophys. Acta 1778, 1814–1838 (2008).

    Article  CAS  Google Scholar 

  3. Rapp, M., Seppälä, S., Granseth, E. & von Heijne, G. Emulating membrane protein evolution by rational design. Science 315, 1282–1284 (2007).

    Article  CAS  Google Scholar 

  4. Schuldiner, S. in Membrane Transport Mechanism (ed. Krämer, R.Z. & Ziegler, C.) 233–248 (Springer, Berlin, 2014).

  5. Fleishman, S.J. et al. Quasi-symmetry in the cryo-EM structure of EmrE provides the key to modeling its transmembrane domain. J. Mol. Biol. 364, 54–67 (2006).

    Article  CAS  Google Scholar 

  6. Ubarretxena-Belandia, I., Baldwin, J.M., Schuldiner, S. & Tate, C.G. Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer. EMBO J. 22, 6175–6181 (2003).

    Article  CAS  Google Scholar 

  7. Chen, Y.J. et al. X-ray structure of EmrE supports dual topology model. Proc. Natl. Acad. Sci. USA 104, 18999–19004 (2007).

    Article  CAS  Google Scholar 

  8. Muth, T.R. & Schuldiner, S. A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE. EMBO J. 19, 234–240 (2000).

    Article  CAS  Google Scholar 

  9. Cho, M.K., Gayen, A., Banigan, J.R., Leninger, M. & Traaseth, N.J. Intrinsic conformational plasticity of native EmrE provides a pathway for multidrug resistance. J. Am. Chem. Soc. 136, 8072–8080 (2014).

    Article  CAS  Google Scholar 

  10. Gayen, A., Banigan, J.R. & Traaseth, N.J. Ligand-induced conformational changes of the multidrug resistance transporter EmrE probed by oriented solid-state NMR spectroscopy. Angew. Chem. Int. Edn Engl. 52, 10321–10324 (2013).

    Article  CAS  Google Scholar 

  11. Morrison, E.A. et al. Antiparallel EmrE exports drugs by exchanging between asymmetric structures. Nature 481, 45–50 (2012).

    Article  CAS  Google Scholar 

  12. Schanda, P. & Brutscher, B. Very fast two-dimensional NMR spectroscopy for real-time investigation of dynamic events in proteins on the time scale of seconds. J. Am. Chem. Soc. 127, 8014–8015 (2005).

    Article  CAS  Google Scholar 

  13. Adam, Y., Tayer, N., Rotem, D., Schreiber, G. & Schuldiner, S. The fast release of sticky protons: kinetics of substrate binding and proton release in a multidrug transporter. Proc. Natl. Acad. Sci. USA 104, 17989–17994 (2007).

    Article  CAS  Google Scholar 

  14. Yerushalmi, H. & Schuldiner, S. An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli. J. Biol. Chem. 275, 5264–5269 (2000).

    Article  CAS  Google Scholar 

  15. Wilks, J.C. & Slonczewski, J.L. pH of the cytoplasm and periplasm of Escherichia coli: rapid measurement by green fluorescent protein fluorimetry. J. Bacteriol. 189, 5601–5607 (2007).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  17. Wang, J. et al. Imaging membrane protein helical wheels. J. Magn. Reson. 144, 162–167 (2000).

    Article  CAS  Google Scholar 

  18. Marassi, F.M. & Opella, S.J. A solid-state NMR index of helical membrane protein structure and topology. J. Magn. Reson. 144, 150–155 (2000).

    Article  CAS  Google Scholar 

  19. Dutta, S., Morrison, E.A. & Henzler-Wildman, K.A. Blocking dynamics of the SMR transporter EmrE impairs efflux activity. Biophys. J. 107, 613–620 (2014).

    Article  CAS  Google Scholar 

  20. deAzevedo, E.R., Bonagamba, T.J. & Schmidt-Rohr, K. Pure-exchange solid-state NMR. J. Magn. Reson. 142, 86–96 (2000).

    Article  CAS  Google Scholar 

  21. Jencks, W.P. Utilization of binding energy and coupling rules for active transport and other coupled vectorial processes. Methods Enzymol. 171, 145–164 (1989).

    Article  CAS  Google Scholar 

  22. Poulsen, B.E., Rath, A. & Deber, C.M. The assembly motif of a bacterial small multidrug resistance protein. J. Biol. Chem. 284, 9870–9875 (2009).

    Article  CAS  Google Scholar 

  23. Amadi, S.T., Koteiche, H.A., Mishra, S. & McHaourab, H.S. Structure, dynamics, and substrate-induced conformational changes of the multidrug transporter EmrE in liposomes. J. Biol. Chem. 285, 26710–26718 (2010).

    Article  CAS  Google Scholar 

  24. Mordoch, S.S., Granot, D., Lebendiker, M. & Schuldiner, S. Scanning cysteine accessibility of EmrE, an H+-coupled multidrug transporter from Escherichia coli, reveals a hydrophobic pathway for solutes. J. Biol. Chem. 274, 19480–19486 (1999).

    Article  CAS  Google Scholar 

  25. Brill, S., Sade-Falk, O., Elbaz-Alon, Y. & Schuldiner, S. Specificity determinants in small multidrug transporters. J. Mol. Biol. 427, 468–477 (2015).

    Article  CAS  Google Scholar 

  26. Banigan, J.R., Gayen, A., Cho, M.K. & Traaseth, N.J. A structured loop modulates coupling between the substrate-binding and dimerization domains in the multidrug resistance transporter EmrE. J. Biol. Chem. 290, 805–814 (2015).

    Article  CAS  Google Scholar 

  27. Rotem, D. & Schuldiner, S. EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry. J. Biol. Chem. 279, 48787–48793 (2004).

    Article  CAS  Google Scholar 

  28. Brill, S., Falk, O.S. & Schuldiner, S. Transforming a drug/H+ antiporter into a polyamine importer by a single mutation. Proc. Natl. Acad. Sci. USA 109, 16894–16899 (2012).

    Article  CAS  Google Scholar 

  29. Son, M.S. et al. Mutagenesis of SugE, a small multidrug resistance protein. Biochem. Biophys. Res. Commun. 312, 914–921 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health (NIH) grant R01AI108889 and start-up funds from New York University (NYU) to N.J.T. M.L. acknowledges support from a Margaret Strauss Kramer Fellowship. The NMR data collected with a cryoprobe at NYU was supported by an NIH S10 grant (OD016343). Data collected at the New York Structural Biology Center was made possible by a grant from NYSTAR. The authors thank R. Turner at the University of Calgary for providing wild-type EmrE in the pMS119EH vector, M.-K. Cho for assistance of the initial NMR experiments, A. Sae Her for assistance in protein purification, and D. Buccella at NYU for use of the spectrofluorometer.

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  1. Anindita Gayen and Maureen Leninger: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemistry, New York University, New York, New York, USA

    Anindita Gayen, Maureen Leninger & Nathaniel J Traaseth

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  1. Anindita Gayen
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Contributions

N.J.T. designed the project. A.G. and M.L. carried out the solution NMR, solid-state NMR, fluorescence experiments and ethidium resistance assays. All authors analyzed the data. N.J.T. wrote the manuscript.

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Correspondence to Nathaniel J Traaseth.

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The authors declare no competing financial interests.

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Supplementary Results, Supplementary Figures 1–14. (PDF 1934 kb)

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Gayen, A., Leninger, M. & Traaseth, N. Protonation of a glutamate residue modulates the dynamics of the drug transporter EmrE. Nat Chem Biol 12, 141–145 (2016). https://doi.org/10.1038/nchembio.1999

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