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Structural basis for the drug extrusion mechanism by a MATE multidrug transporter

Nature volume 496, pages 247–251 (2013)Cite this article

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An Author Correction to this article was published on 30 January 2020

Abstract

Multidrug and toxic compound extrusion (MATE) family transporters are conserved in the three primary domains of life (Archaea, Bacteria and Eukarya), and export xenobiotics using an electrochemical gradient of H+ or Na+ across the membrane1,2. MATE transporters confer multidrug resistance to bacterial pathogens3,4,5,6 and cancer cells7, thus causing critical reductions in the therapeutic efficacies of antibiotics and anti-cancer drugs, respectively. Therefore, the development of MATE inhibitors has long been awaited in the field of clinical medicine8,9. Here we present the crystal structures of the H+-driven MATE transporter from Pyrococcus furiosus in two distinct apo-form conformations, and in complexes with a derivative of the antibacterial drug norfloxacin and three in vitro selected thioether-macrocyclic peptides, at 2.1–3.0 Å resolutions. The structures, combined with functional analyses, show that the protonation of Asp 41 on the amino (N)-terminal lobe induces the bending of TM1, which in turn collapses the N-lobe cavity, thereby extruding the substrate drug to the extracellular space. Moreover, the macrocyclic peptides bind the central cleft in distinct manners, which correlate with their inhibitory activities. The strongest inhibitory peptide that occupies the N-lobe cavity may pave the way towards the development of efficient inhibitors against MATE transporters.

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Figure 1: Overall structures of PfMATE.
Figure 2: Mutational analyses of PfMATE.
Figure 3: Complex structure of PfMATE and drug substrate.
Figure 4: Complex structures with the macrocyclic peptides.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates and structure factors for the P. furiosus apo MATE (‘straight’ and ‘bent’ conformations), P26A mutant, Br-NRF-bound MATE and peptide-bound MATEs have been deposited in the Protein Data Bank, under the accession numbers 3VVN, 3VVO, 3W4T, 3VVP, 3VVQ, 3VVR and 3VVS, respectively.

References

  1. Brown, M. H., Paulsen, I. T. & Skurray, R. A. The multidrug efflux protein NorM is a prototype of a new family of transporters. Mol. Microbiol. 31, 394–395 (1999)

    Article  CAS  Google Scholar 

  2. He, G. X. et al. An H+-coupled multidrug efflux pump, PmpM, a member of the MATE family of transporters, from Pseudomonas aeruginosa. J. Bacteriol. 186, 262–265 (2004)

    Article  CAS  Google Scholar 

  3. Kaatz, G. W., McAleese, F. & Seo, S. M. Multidrug resistance in Staphylococcus aureus due to overexpression of a novel multidrug and toxin extrusion (MATE) transport protein. Antimicrob. Agents Chemother. 49, 1857–1864 (2005)

    Article  CAS  Google Scholar 

  4. McAleese, F. et al. A novel MATE family efflux pump contributes to the reduced susceptibility of laboratory-derived Staphylococcus aureus mutants to tigecycline. Antimicrob. Agents Chemother. 49, 1865–1871 (2005)

    Article  CAS  Google Scholar 

  5. Tsuda, M. et al. Oppositely directed H+ gradient functions as a driving force of rat H+/organic cation antiporter MATE1. Am. J. Physiol. 292, F593–F598 (2007)

    CAS  Google Scholar 

  6. Becker, M. L. et al. Genetic variation in the multidrug and toxin extrusion 1 transporter protein influences the glucose-lowering effect of metformin in patients with diabetes: a preliminary study. Diabetes 58, 745–749 (2009)

    Article  CAS  Google Scholar 

  7. Minematsu, T. & Giacomini, K. M. Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins. Mol. Cancer Ther. 10, 531–539 (2011)

    Article  CAS  Google Scholar 

  8. Ito, S. et al. Potent and specific inhibition of mMate1-mediated efflux of type I organic cations in the liver and kidney by pyrimethamine. J. Pharmacol. Exp. Ther. 333, 341–350 (2010)

    Article  CAS  Google Scholar 

  9. Kusuhara, H. et al. Effects of a MATE protein inhibitor, pyrimethamine, on the renal elimination of metformin at oral microdose and at therapeutic dose in healthy subjects. Clin. Pharmacol. Ther. 89, 837–844 (2011)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  11. 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)

    Article  ADS  CAS  Google Scholar 

  12. Wasaznik, A., Grinholc, M. & Bielawski, K. P. Active efflux as the multidrug resistance mechanism. Postepy Hig. Med. Dosw. 63, 123–133 (2009)

    Google Scholar 

  13. Morita, Y. et al. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli. Antimicrob. Agents Chemother. 42, 1778–1782 (1998)

    Article  CAS  Google Scholar 

  14. Morita, Y., Kataoka, A., Shiota, S., Mizushima, T. & Tsuchiya, T. NorM of Vibrio parahaemolyticus is an Na+-driven multidrug efflux pump. J. Bacteriol. 182, 6694–6697 (2000)

    Article  CAS  Google Scholar 

  15. Otsuka, M. et al. Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus. J. Bacteriol. 187, 1552–1558 (2005)

    Article  CAS  Google Scholar 

  16. He, X. et al. Structure of a cation-bound multidrug and toxic compound extrusion transporter. Nature 467, 991–994 (2010)

    Article  ADS  CAS  Google Scholar 

  17. Law, C. J., Maloney, P. C. & Wang, D. N. Ins and outs of major facilitator superfamily antiporters. Annu. Rev. Microbiol. 62, 289–305 (2008)

    Article  CAS  Google Scholar 

  18. Cherezov, V. Lipidic cubic phase technologies for membrane protein structural studies. Curr. Opin. Struct. Biol. 21, 559–566 (2011)

    Article  CAS  Google Scholar 

  19. Hipolito, C. J. & Suga, H. Ribosomal production and in vitro selection of natural product-like peptidomimetics: the FIT and RaPID systems. Curr. Opin. Chem. Biol. 16, 196–203 (2012)

    Article  CAS  Google Scholar 

  20. Hayashi, Y., Morimoto, J. & Suga, H. In vitro selection of anti-Akt2 thioether-macrocyclic peptides leading to isoform-selective inhibitors. ACS Chem. Biol. 7, 607–613 (2012)

    Article  CAS  Google Scholar 

  21. Yamagishi, Y. et al. Natural product-like macrocyclic N-methyl-peptide inhibitors against a ubiquitin ligase uncovered from a ribosome-expressed de novo library. Chem. Biol. 18, 1562–1570 (2011)

    Article  CAS  Google Scholar 

  22. Morimoto, J., Hayashi, Y. & Suga, H. Discovery of macrocyclic peptides armed with a mechanism-based warhead: isoform-selective inhibition of human deacetylase SIRT2. Angew. Chem. Int. Ed. 51, 3423–3427 (2012)

    Article  CAS  Google Scholar 

  23. Altschul, S. F. et al. Protein database searches using compositionally adjusted substitution matrices. FEBS J. 272, 5101–5109 (2005)

    Article  CAS  Google Scholar 

  24. Koga, H., Itoh, A., Murayama, S., Suzue, S. & Irikura, T. Structure-activity relationships of antibacterial 6,7- and 7,8-disubstituted 1-alkyl-1,4-dihydro-4-oxoquinoline-3-carboxylic acids. J. Med. Chem. 23, 1358–1363 (1980)

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  26. Flot, D. et al. The ID23–2 structural biology microfocus beamline at the ESRF. J. Synchrotron Radiat. 17, 107–118 (2010)

    Article  CAS  Google Scholar 

  27. Tsujimoto, K., Semadeni, M., Huflejt, M. & Packer, L. Intracellular pH of halobacteria can be determined by the fluorescent dye 2′, 7′-bis(carboxyethyl)-5(6)-carboxyfluorescein. Biochem. Biophys. Res. Commun. 155, 123–129 (1988)

    Article  CAS  Google Scholar 

  28. Li, X. Z., Poole, K. & Nikaido, H. Contributions of MexAB-OprM and an EmrE homolog to intrinsic resistance of Pseudomonas aeruginosa to aminoglycosides and dyes. Antimicrob. Agents Chemother. 47, 27–33 (2003)

    Article  CAS  Google Scholar 

  29. Hirata, K. et al. New micro-beam beamline at SPring-8, targeting at protein micro-crystallography. AIP Conf. Proc. 1234, 901–904 (2010)

    Article  ADS  CAS  Google Scholar 

  30. Xu, H., Smith, A. B., Sahinidis, N. V. & Weeks, C. M. SnB version 2.3: triplet sieve phasing for centrosymmetric structures. J. Appl. Cryst. 41, 644–646 (2008)

    Article  CAS  Google Scholar 

  31. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007)

    CAS  Google Scholar 

  32. Eswar, N. et al. Comparative protein structure modeling using MODELLER. Curr. Protoc. Protein Sci. 2, 2.9.1–2.9.31 (2007)

    Article  Google Scholar 

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

    Article  Google Scholar 

  34. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  35. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  36. Smart, O. S. et al. Exploiting structure similarity in refinement: automated NCS and target-structure restraints in BUSTER. Acta Crystallogr. D 68, 368–380 (2012)

    Article  CAS  Google Scholar 

  37. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  39. Zwart, P. H. et al. Automated structure solution with the PHENIX suite. Methods Mol. Biol. 426, 419–435 (2008)

    Article  CAS  Google Scholar 

  40. Ito, K., Ebihara, K., Uno, M. & Nakamura, Y. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis. Proc. Natl Acad. Sci. USA 93, 5443–5448 (1996)

    Article  ADS  CAS  Google Scholar 

  41. Martinac, B., Buechner, M., Delcour, A. H., Adler, J. & Kung, C. Pressure-sensitive ion channel in Escherichia coli. Proc. Natl Acad. Sci. USA 84, 2297–2301 (1987)

    Article  ADS  CAS  Google Scholar 

  42. Kuo, M. M., Saimi, Y., Kung, C. & Choe, S. Patch clamp and phenotypic analyses of a prokaryotic cyclic nucleotide-gated K+ channel using Escherichia coli as a host. J. Biol. Chem. 282, 24294–24301 (2007)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to the beam-line staff at BL32XU of SPring-8 for assistance in data collection (proposals 2011B1062, 2012A1087 and 2012B1161), and the RIKEN BioResource Center (Ibaraki, Japan) for providing the P. furiosus genomic DNA. We are also grateful to N. Dohmae and Y. Sugita (RIKEN Advanced Science Institute, Japan) for discussions. This work was supported by the Japan Society for the Promotion of Science (JSPS) through its ‘Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST program)’ to O.N.; by the Core Research for Evolutional Science and Technology Program ‘The Creation of Basic Medical Technologies to Clarify and Control the Mechanisms Underlying Chronic Inflammation’ of Japan Science and Technology Agency to O.N.; and by a Grant-in-Aid for Scientific Research (S) (24227004) and a Grant-in-Aid for Young Scientists (A) (22687007) from MEXT to O.N. and R.I., respectively. This work was also supported by a JSPS Grant-in-Aid for the Specially Promoted Research (21000005) and MEXT Platform for Drug Discovery, Informatics, and Structural Life Science to H.S., and a Grant-in-Aid for JSPS post-doctoral fellows to C.J.H. (P11344).

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Author notes

  1. Yoshiki Tanaka

    Present address: Present address: Department of Medical Chemistry and Cell Biology, Faculty of Medicine, Kyoto University, Konoe-cho, Yoshida, Sakyo-ku, Kyoto, 606-8501, Japan.,

Authors and Affiliations

  1. RIKEN Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan,

    Yoshiki Tanaka, Hideaki E. Kato, Motoyuki Hattori, Kaoru Kumazaki, Tomoya Tsukazaki, Ryuichiro Ishitani & Osamu Nureki

  2. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan,

    Yoshiki Tanaka, Hideaki E. Kato, Motoyuki Hattori, Kaoru Kumazaki, Tomoya Tsukazaki, Ryuichiro Ishitani & Osamu Nureki

  3. Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Bunkyo-ku, Tokyo 113-0033, Japan,

    Christopher J. Hipolito, Takashi Higuchi, Takayuki Katoh & Hiroaki Suga

  4. Department of Bioengineering Sciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan,

    Andrés D. Maturana

  5. Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan,

    Koichi Ito

  6. Department of Genome Applied Microbiology, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, 700-8530, Tsushima, Japan

    Teruo Kuroda

  7. Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan,

    Motoyuki Hattori & Tomoya Tsukazaki

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Contributions

Y.T. expressed and purified PfMATE for crystallization, collected the diffraction data, solved the structures and made the mutants for functional analyses. C.J.H. performed selections, syntheses and inhibition assays of macrocyclic peptides. A.D.M. performed the fluorescence analysis. K.I. performed growth complementation tests. T.K. performed drug susceptibility tests. T.H. synthesized Br-NRF. K.K. and H.E.K. assisted with data collection. M.H. and T.T. contributed to the early stage of the project. Y.T., C.J.H., H.E.K., R.I. and O.N. wrote the manuscript. H.S. and O.N. directed and supervised all of the research.

Corresponding authors

Correspondence to Hiroaki Suga or Osamu Nureki.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains a Supplementary Discussion, Supplementary Tables 1-2, Supplementary Figures 1-10 and Supplementary References. (PDF 5096 kb)

Proton-dependent drug extrusion by MATE

The structural transition between the “straight” and “bent” conformations, viewed from the extracellular and membrane sides. (MP4 3137 kb)

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Tanaka, Y., Hipolito, C., Maturana, A. et al. Structural basis for the drug extrusion mechanism by a MATE multidrug transporter. Nature 496, 247–251 (2013). https://doi.org/10.1038/nature12014

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