Covalently Linked Trimers of RND (Resistance-Nodulation-Division) Efflux Transporters to Study Their Mechanism of Action: Escherichia coli AcrB Multidrug Exporter as an Example

  • Hiroshi Nikaido
Part of the Methods in Molecular Biology book series (MIMB, volume 1700)


Transporters undergo large conformational changes in their functional cycle. RND (Resistance-Nodulation-Division) family efflux transporters usually exist as homotrimers, and each protomer was proposed to undergo a cycle of conformational changes in succession so that at any given time the trimer would contain three protomers of different conformations, the functionally rotating mechanism of transport. This mechanism implies that the inactivation of one protomer among three will inactivate the entire trimeric ensemble by blocking the functional rotation. We describe a biochemical approach to test this prediction by first producing a giant protein in which the three protomers of Escherichia coli AcrB efflux pump are covalently linked together through linker sequences, and then testing for its function by inactivation of a single protomer unit. Inactivation can be done permanently by mutating a residue involved in proton relay, or in “real time” by using a protein in which one protomer contains two Cys residues on both sides of the large cleft in the periplasmic domain and then by rapidly inactivating this protomer with a methanethiosulfonate cross-linker.

Key words

RND family transporters Antibiotics Drug efflux Disulfide bonds Methanethiosulfonate AcrB Proton relay Functionally rotating mechanism 



This work was supported by a grant from the U.S. Public Health Service (AI-009644). I thank Y. Takatsuka for supplying the details and for suggestions.


  1. 1.
    Li X-Z, Plesiat P, Nikaido H (2015) The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev 28:337–418CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Murakami S, Nakashima R, Yamashita E et al (2002) Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419:587–593CrossRefPubMedGoogle Scholar
  3. 3.
    Murakami S, Nakashima R, Yamashita E et al (2006) Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443:173–179CrossRefPubMedGoogle Scholar
  4. 4.
    Seeger MA, Schiefner A, Eicher T et al (2006) Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313:1295–1298CrossRefPubMedGoogle Scholar
  5. 5.
    Sennhauser G, Amstutz P, Briand C et al (2007) Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors. PLoS Biol e7:5Google Scholar
  6. 6.
    Cha HJ, Pos KM (2014) Cooperative transport mechanism and proton-coupling in the multidrug efflux transporter complex AcrAB-TolC. In: Kramer R, Ziegler C (eds) Membrane transport mechanisms: 3D structure and beyond. Springer, Heidelberg, pp 207–232CrossRefGoogle Scholar
  7. 7.
    Takatsuka Y, Nikaido H (2009) Covalently linked trimer of the AcrB multidrug efflux pump provides support for the functional rotating mechanism. J Bacteriol 191:1729–1737CrossRefPubMedGoogle Scholar
  8. 8.
    Kinana AD, Vargiu AV, Nikaido H (2013) Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB. Biochemistry 52:8342–8351CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Kim HS, Nikaido H (2012) Different functions of MdtB and MdtC subunits in the heterotrimeric efflux transporter MdtB(2)C complex of Escherichia coli. Biochemistry 51:4188–4197CrossRefPubMedGoogle Scholar
  10. 10.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  11. 11.
    Rietsch A, Belin D, Martin N et al (1996) An in vivo pathway for disulfide bond isomerization in Escherichia coli. Proc Natl Acad Sci U S A 93:13048–13053CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Okusu H, Ma D, Nikaido H (1996) AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli multiple-antibiotic-resistance (Mar) mutants. J Bacteriol 178:306–308CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Takatsuka Y, Nikaido H (2007) Site-directed disulfide cross-linking shows that cleft flexibility in the periplasmic domain is needed for the multidrug efflux pump AcrB of Escherichia coli. J Bacteriol 189:8677–8684CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Churchward G, Belin D, Nagamine Y (1984) A pSC101-derived plasmid which shows no sequence homology to other commonly used cloning vectors. Gene 31:165–171CrossRefPubMedGoogle Scholar
  15. 15.
    Ma D, Cook DN, Alberti M et al (1993) Molecular cloning and characterization of acrA and acrE genes of Escherichia coli. J Bacteriol 175:6299–6313CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Takatsuka Y, Nikaido H (2006) Threonine-978 in the transmembrane segment of the multidrug efflux pump AcrB of Escherichia coli is crucial for drug transport as a probable component of the proton relay network. J Bacteriol 188:7284–7289CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Schagger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–231CrossRefPubMedGoogle Scholar
  18. 18.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  19. 19.
    Zgurskaya HI, Nikaido H (1999) Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proc Natl Acad Sci U S A 96:7190–7195CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Takatsuka Y, Nikaido H (2010) Site-directed disulfide cross-linking to probe conformational changes of a transporter during its functional cycle: Escherichia coli AcrB multidrug exporter as an example. Methods Mol Biol 634:343–354CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Su CC, Li M, Gu R et al (2006) Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway. J Bacteriol 188:7290–7296CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Nikaido H (1994) Isolation of outer membranes. Methods Enzymol 235:225–234CrossRefPubMedGoogle Scholar
  23. 23.
    Elkins CA, Nikaido H (2002) Substrate specificity of the RND-type multidrug efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large periplasmic loops. J Bacteriol 184:6490–6498CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Bryson V, Szybalski W (1952) Microbial selection. Science 116:45–51CrossRefGoogle Scholar
  25. 25.
    Goldberg AL (2003) Protein degradation and protection against misfolded or damaged proteins. Nature 426:895–899CrossRefPubMedGoogle Scholar
  26. 26.
    Kenyon GL, Bruice TW (1977) Novel sulfhydryl reagents. Methods Enzymol 47:407–430CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Department of Molecular and Cell BiologyUniversity of California, BerkeleyCAUSA

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