Advertisement

Crystallographic Analysis of Drug and Inhibitor-Binding Structure of RND-Type Multidrug Exporter AcrB in Physiologically Relevant Asymmetric Crystals

  • Ryosuke Nakashima
  • Keisuke Sakurai
  • Akihito Yamaguchi
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)

Abstract

Xenobiotic extruding pumps have recently been known to be widely distributed in living organisms from mammalian to bacteria as a host-defense mechanism in cellular level. These pumps not only confer multidrug resistance of cancer cells and pathogenic bacteria but also cause hereditary diseases through the mutation. Our purposes are to elucidate the molecular structures and mechanisms of these xenobiotic exporters.

We had succeeded to determine the crystal structure of bacterial major multidrug exporter AcrB at 3.5 Å resolution (Murakami et al., Nature 419:587–593, 2002) and elucidated the structural bases of substrate recognition that the pump recognize the places and thus act as a “membrane vacuum cleaner.” After that we also determined the crystal structure of the drug-binding form of AcrB in space group C2 in which asymmetric unit contains structurally asymmetric homo-trimer of AcrB (Murakami et al., Nature 443:173–179, 2006; Nakashima et al., Nature 480:565–569, 2011; Nakashima et al., Nature 500:120–126, 2013). Analyses revealed the existence of a specific mechanism to recognize numerous substrates that the multisite binding is the base of multidrug recognition rather than induced-fit, and functional-rotation mechanism in which three monomers undergo a strictly coordinated sequential conformational change cycle of access, binding, and extrusion. Determination of physiological asymmetric AcrB structure was crucially important to understand these transport mechanisms.

Key words

Multidrug exporter AcrB RND family Crystallization X-ray Crystal structure Functional-rotation mechanism Peristaltic pump mechanism 

Notes

Acknowledgements

This work was supported by CREST, JST.

References

  1. 1.
    McMurry L, Petrucci RE Jr, Levy SB (1980) Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci U S A 77:3974–3977CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Fujihira E, Tamura N, Yamaguchi A (2002) Membrane topology of a multidrug efflux transporter, AcrB, in Escherichia coli. J Biochem (Tokyo) 131:145–151Google Scholar
  3. 3.
    Murakami S, Nakashima R, Yamashita E, Yamaguchi A (2002) Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419:587–593CrossRefPubMedGoogle Scholar
  4. 4.
    Veesler D, Blangy S, Cambillau C, Sciara G (2008) There is a baby in the bath water: AcrB contamination is a major problem in membrane-protein crystallization. Acta Crystallogr Sect F Struct Biol Cryst Commun 64:880–885CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Nakashima R, Sakurai K, Yamasaki S, Hayashi K, Nagata C, Hoshino K, Onodera Y, Nishino K, Yamaguchi A (2013) Structural basis for the inhibition of bacterial multidrug exporters. Nature 500:120–126CrossRefGoogle Scholar
  6. 6.
    Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol 276:307–326CrossRefGoogle Scholar
  7. 7.
    Collaborative Computational Project (1994) Number 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50:760–763CrossRefGoogle Scholar
  8. 8.
    Vagin A, Teplyakov A (1997) MOLREP: an automated program for molecular replacement. J Appl Crystallogr 30:1022–1025CrossRefGoogle Scholar
  9. 9.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D 60:2126–2132CrossRefPubMedGoogle Scholar
  10. 10.
    Brunger AT (2007) Version 1.2 of the crystallography and NMR system. Nat Protoc 2:2728–2733CrossRefPubMedGoogle Scholar
  11. 11.
    Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D 53:240–255CrossRefPubMedGoogle Scholar
  12. 12.
    Yu EW, McDermott G, Zgurskaya HI, Nikaido H, Koshland DE Jr (2003) Structural basis of multiple drug binding capacity of the AcrB multidrug efflux pump. Science 300:976–980CrossRefPubMedGoogle Scholar
  13. 13.
    Pos KM, Schiefner A, Seeger MA, Diederichs K (2004) Crystallographic analysis of AcrB. FEBS Lett 564:333–339CrossRefPubMedGoogle Scholar
  14. 14.
    Das D, Xu QS, Lee JY, Ankoudinova I, Huang C, Lou Y, DeGiovanni A, Kim R, Kim SH (2007) Crystal structure of the multidrug efflux transporter AcrB at 3.1 Å resolution reveals the N-terminal region with conserved amino acids. J Struct Biol 158:494–502CrossRefPubMedGoogle Scholar
  15. 15.
    Drew D, Klepsch MM, Newstead S, Flaig R, De Gier JW, Iwata S, Beis K (2008) The structure of the efflux pump AcrB in complex with bile acid. Mol Membr Biol 25:677–682CrossRefPubMedGoogle Scholar
  16. 16.
    Hung LW, Kim HB, Murakami S, Gupta G, Kim CY, Terwilliger TC (2013) Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution. J Struct Funct Genom 14:71–75CrossRefGoogle Scholar
  17. 17.
    Törnroth-Horsefield S, Gourdon P, Horsefield R, Brive L, Yamamoto N, Mori H, Snijder A, Neutze R (2007) Crystal structure of AcrB in complex with a single transmembrane subunit reveals another twist. Structure 15:1663–1673CrossRefPubMedGoogle Scholar
  18. 18.
    Du D, Wang Z, James NR, Voss JE, Klimont E, Ohene-Agyei T, Venter H, Chiu W, Luisi BF (2014) Structure of the AcrAB-TolC multidrug efflux pump. Nature 509:512–515CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Long F, Su CC, Zimmermann MT, Boyken SE, Rajashankar KR, Jernigan RL, Yu EW (2010) Crystal structures of the CusA efflux pump suggest methionine-mediated metal transport. Nature 467:484–488CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Su CC, Long F, Zimmermann MT, Rajashankar KR, Jernigan RL, Yu EW (2011) Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli. Nature 470:558–562CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Lei HT, Chou TH, Su CC, Bolla JR, Kumar N, Radhakrishnan A, Long F, Delmar JA, Do SV, Rajashankar KR, Shafer WM, Yu EW (2015) Crystal structure of the Neisseria gonorrhoeae MtrD inner membrane multidrug efflux pump. PLoS One 6:e97475Google Scholar
  22. 22.
    Murakami S, Nakashima R, Yamashita E, Matsumoto T, Yamaguchi A (2006) Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443:173–179CrossRefPubMedGoogle Scholar
  23. 23.
    Seeger MA, Schiefner A, Eicher T, Verrey F, Diederichs K, Pos KM (2006) Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313:1295–1298CrossRefPubMedGoogle Scholar
  24. 24.
    Sennhauser G, Amstutz P, Briand C, Storchenegger O, Grütter MG (2006) Drug export pathway of multidrug exporter AcrB revealed by Darpin inhibitors. PLoS Biol 5:e7CrossRefPubMedCentralGoogle Scholar
  25. 25.
    Sennhauser G, Bukowska MA, Briand C, Grütter MG (2009) Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa. J Mol Biol 389:134–145CrossRefPubMedGoogle Scholar
  26. 26.
    Pak JE, Ekendé EN, Kifle EG, O’Connell JD 3rd, De Angelis F, Tessema MB, Derfoufi KM, Robles-Colmenares Y, Robbins RA, Goormaghtigh E, Vandenbussche G, Stroud RM (2013) Structures of intermediate transport states of ZneA, a Zn(II)/proton antiporter. Proc Natl Acad Sci U S A 110:18484–18489CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Nakashima R, Sakurai K, Yamasaki S, Nishino K, Yamaguchi A (2011) Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 480:565–569PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  • Ryosuke Nakashima
    • 1
  • Keisuke Sakurai
    • 1
  • Akihito Yamaguchi
    • 1
  1. 1.Department of Cell Membrane Biology, Institute of Scientific and Industrial ResearchOsaka UniversityIbarakiJapan

Personalised recommendations