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Licensed Unlicensed Requires Authentication Published by De Gruyter June 9, 2015

The assembly and disassembly of the AcrAB-TolC three-component multidrug efflux pump

Reinke Tobias Müller and Klaas Martinus Pos
From the journal Biological Chemistry


In Gram-negative bacteria, tripartite efflux pumps, like AcrAB-TolC from Escherichia coli, play a prominent role in the resistance against multiple antibiotics. Transport of the drugs across the outer membrane and its coupling to the electrochemical gradient is dependent on the presence of all three components. As the activity of the E. coli AcrAB-TolC efflux pump is dependent on both the concentration of substrates and the extent of the electrochemical gradient across the inner membrane, the dynamics of tripartite pump assembly and disassembly might be crucial for effective net transport of drugs towards the outside of the cell.

Corresponding author: Klaas Martinus Pos, Institute of Biochemistry, Goethe University Frankfurt, D-60438 Frankfurt/Main, Germany, e-mail:


Many of the ideas described in this review arose during the workshop ‘Toward an integrated understanding of drug resistance’ in Santa Fe, NM, USA on 18–20 February 2015. I would like to thank all the colleagues present at that occasion for their open discussion spirit. Work in the Pos lab is supported by the German Research Foundation (SFB 807, Transport and Communication across Biological Membranes and FOR2251, Adaptation and persistence of the emerging pathogen Acinetobacter baumannii), the DFG-EXC115 (Cluster of Excellence Macromolecular Complexes at the Goethe University Frankfurt), Innovative Medicines Initiative Joint Undertaking Project Translocation (IMI-Translocation), EU Marie Curie Actions ITN, Human Frontiers Science Program (HFSP) and the German-Israeli Foundation (GIF).


Boyer, P.D. (1997). The ATP synthase – a splendid molecular machine. Annu. Rev. Biochem. 66, 717–749.Search in Google Scholar

Cha, H. and Pos, K.M. (2014). Membrane Transport Mechanism (Berlin, Heidelberg: Springer Berlin Heidelberg).Search in Google Scholar

Cha, H.J., Müller, R.T., and Pos, K.M. (2014). Switch-loop flexibility affects transport of large drugs by the promiscuous AcrB multidrug efflux transporter. Antimicrob. Agents Chemother. 58, 4767–4772.Search in Google Scholar

Cherepanov, D.A., Feniouk, B.A., Junge, W., and Mulkidjanian, A.Y. (2003). Low dielectric permittivity of water at the membrane interface: effect on the energy coupling mechanism in biological membranes. Biophys. J. 85, 1307–1316.Search in Google Scholar

Cherepanov, D.A., Junge, W., and Mulkidjanian, A.Y. (2004). Proton transfer dynamics at the membrane/water interface: dependence on the fixed and mobile pH buffers, on the size and form of membrane particles, and on the interfacial potential barrier. Biophys. J. 86, 665–680.Search in Google Scholar

Du, D., Wang, Z., James, N.R., Voss, J.E., Klimont, E., Ohene-Agyei, T., Venter, H., Chiu, W., and Luisi, B.F. (2014). Structure of the AcrAB-TolC multidrug efflux pump. Nature 509, 512–515.Search in Google Scholar

Du, D., van Veen, H.W., and Luisi, B.F. (2015a). Assembly and operation of bacterial tripartite multidrug efflux pumps. Trends Microbiol. 23, 311–319.Search in Google Scholar

Du, D., Voss, J., Wang, Z., Chiu, W., and Luisi, B.F. (2015b). The pseudo-atomic structure of an RND-type tripartite multidrug efflux pump. Biol. Chem. 396, 1073–1082.Search in Google Scholar

Eicher, T., Cha, H.J., Seeger, M.A., Brandstätter, L., El-Delik, J., Bohnert, J.A, Kern, W.V, Verrey, F., Grütter, M.G., Diederichs, K., et al. (2012). Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proc. Natl. Acad. Sci. USA 109, 5687–5692.Search in Google Scholar

Eicher, T., Seeger, M.A., Anselmi, C., Zhou, W., Brandstätter, L., Verrey, F., Diederichs, K., Faraldo-Gómez, J.D., and Pos, K.M. (2014). Coupling of remote alternating-access transport mechanisms for protons and substrates in the multidrug efflux pump AcrB. Elife 3, e03145.Search in Google Scholar

Fischer, N. and Kandt, C. (2011). Three ways in, one way out: water dynamics in the trans-membrane domains of the inner membrane translocase AcrB. Proteins 79, 2871–2885.Search in Google Scholar

Hung, L.-W., Kim, H.-B., Murakami, S., Gupta, G., Kim, C.-Y., and Terwilliger, T.C. (2013). Crystal structure of AcrB complexed with linezolid at 3.5 Å resolution. J. Struct. Funct. Genomics 14, 71–75.Search in Google Scholar

Janganan, T.K., Bavro, V.N., Zhang, L., Borges-Walmsley, M.I., and Walmsley, A.R. (2013). Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump. Mol. Microbiol. 88, 590–602.Search in Google Scholar

Kashket, E.R. (1985). The proton motive force in bacteria: a critical assessment of methods. Annu. Rev. Microbiol. 39, 219–242.Search in Google Scholar

Kinana, A.D., Vargiu, A.V, and Nikaido, H. (2013). Some ligands enhance the efflux of other ligands by the Escherichia coli multidrug pump AcrB. Biochemistry 52, 8342–8351.Search in Google Scholar

Kralj, J.M., Hochbaum, D.R., Douglass, A.D., and Cohen, A.E. (2011). Electrical spiking in Escherichia coli probed with a fluorescent voltage-indicating protein. Science 333, 345–348.Search in Google Scholar

Lim, S.P. and Nikaido, H. (2010). Kinetic parameters of efflux of penicillins by the multidrug efflux transporter AcrAB-TolC of Escherichia coli. Antimicrob. Agents Chemother. 54, 1800–1806.Search in Google Scholar

Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002). Crystal structure of bacterial multidrug efflux transporter AcrB. Nature 419, 587–593.Search in Google Scholar

Murakami, S., Nakashima, R., Yamashita, E., Matsumoto, T., and Yamaguchi, A. (2006). Crystal structures of a multidrug transporter reveal a functionally rotating mechanism. Nature 443, 173–179.Search in Google Scholar

Nagano, K. and Nikaido, H. (2009). Kinetic behavior of the major multidrug efflux pump AcrB of Escherichia coli. Proc. Natl. Acad. Sci. USA 106, 5854–5858.Search in Google Scholar

Nakashima, R., Sakurai, K., Yamasaki, S., Nishino, K., and Yamaguchi, A. (2011). Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 480, 565–569.Search in Google Scholar

Nakashima, R., Sakurai, K., Yamasaki, S., Hayashi, K., Nagata, C., Hoshino, K., Onodera, Y., Nishino, K., and Yamaguchi, A. (2013). Structural basis for the inhibition of bacterial multidrug exporters. Nature 500, 102–106.Search in Google Scholar

Nikaido, H. (1996). Multidrug efflux pumps of gram-negative bacteria. J. Bacteriol. 178, 5853–5859.Search in Google Scholar

Ntsogo Enguene, V.Y., Verchère, A., Phan, G., Broutin, I., and Picard, M. (2015). Catch me if you can: a biotinylated proteoliposome affinity assay for the investigation of assembly of the MexA-MexB-OprM efflux pump from Pseudomonas aeruginosa. Front. Microbiol. 6. doi: 10.3389/fmicb.2015.00541.Search in Google Scholar

Ohene-Agyei, T., Lea, J.D., and Venter, H. (2012). Mutations in MexB that affect the efflux of antibiotics with cytoplasmic targets. FEMS Microbiol. Lett. 333, 20–27.Search in Google Scholar

Piddock, L.J. (2006). Clinically relevant chromosomally encoded multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev. 19, 382–402.Search in Google Scholar

Piddock, L.J.V (2014). Understanding the basis of antibiotic resistance: a platform for drug discovery. Microbiology 160, 2366–2373.Search in Google Scholar

Pos, K.M. (2009). Drug transport mechanism of the AcrB efflux pump. Biochim. Biophys. Acta. 1794, 782–793.Search in Google Scholar

Ruggerone, P., Murakami, S., Pos, K.M., Vargiu, A.V, Pos, K.M., and Vargiu, A.V (2013). RND efflux pumps: structural information translated into function and inhibition mechanisms. Curr. Top. Med. Chem. 13, 3079–3100.Search in Google Scholar

Seeger, M.A, Schiefner, A., Eicher, T., Verrey, F., Diederichs, K., and Pos, K.M. (2006). Structural asymmetry of AcrB trimer suggests a peristaltic pump mechanism. Science 313, 1295–1298.Search in Google Scholar

Seeger, M.A., von Ballmoos, C., Verrey, F., and Pos, K.M. (2009). Crucial role of Asp408 in the proton translocation pathway of multidrug transporter AcrB: evidence from site-directed mutagenesis and carbodiimide labeling. Biochemistry 48, 5801–5812.Search in Google Scholar

Su, C.-C.C., Li, M., Gu, R., Takatsuka, Y., McDermott, G., Nikaido, H., and Yu, E.W. (2006). Conformation of the AcrB multidrug efflux pump in mutants of the putative proton relay pathway. J. Bacteriol. 188, 7290–7296.Search in Google Scholar

Tikhonova, E.B., Yamada, Y., and Zgurskaya, H.I. (2011). Sequential mechanism of assembly of multidrug efflux pump AcrAB-TolC. Chem. Biol. 18, 454–463.Search in Google Scholar

Vargiu, A.V. and Nikaido, H. (2012). Multidrug binding properties of the AcrB efflux pump characterized by molecular dynamics simulations. Proc. Natl. Acad. Sci. USA 109, 20637–20642.Search in Google Scholar

Verchère, A., Dezi, M., Broutin, I., and Picard, M. (2014). In vitro investigation of the MexAB efflux pump from Pseudomonas aeruginosa. J. Vis. Exp. e50894.Search in Google Scholar

Verchère, A., Dezi, M., Adrien, V., Broutin, I., and Picard, M. (2015). In vitro transport activity of the fully assembled MexAB-OprM efflux pump from Pseudomonas aeruginosa. Nat. Commun. 6, 6890.Search in Google Scholar

Yao, X.Q., Kenzaki, H., Murakami, S., and Takada, S. (2010). Drug export and allosteric coupling in a multidrug transporter revealed by molecular simulations. Nat. Commun. 1, 117.Search in Google Scholar

Zgurskaya, H.I. and Nikaido, H. (1999a). Bypassing the periplasm: reconstitution of the AcrAB multidrug efflux pump of Escherichia coli. Proc. Natl. Acad. Sci. USA 96, 7190–7195.Search in Google Scholar

Zgurskaya, H.I. and Nikaido, H. (1999b). AcrA is a highly asymmetric protein capable of spanning the periplasm. J. Mol. Biol. 285, 409–420.Search in Google Scholar

Zgurskaya, H.I., Weeks, J.W., Ntreh, A.T., Nickels, L.M., and Wolloscheck, D. (2015). Mechanism of coupling drug transport reactions located in two different membranes. Front. Microbiol. 6, 100.Search in Google Scholar

Received: 2015-3-31
Accepted: 2015-6-3
Published Online: 2015-6-9
Published in Print: 2015-9-1

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