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Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB

Nature Structural & Molecular Biology volume 15, pages 199–205 (2008)Cite this article

Abstract

The AcrA–AcrB–TolC complex is the major multidrug efflux pump in Escherichia coli. The asymmetric structure of the trimeric inner-membrane component AcrB implies functional rotation of the monomers and a peristaltic mode of drug efflux. This mechanism suggests the occurrence of conformational changes in the periplasmic pore domain through the movements of subdomains during cycling of the monomers through the different states loose (L), tight (T) and open (O). We introduced cysteines at the interfaces of potentially moving subdomains, leading to disulfide bond formation as quantified by alkylation of free cysteines and MALDI-TOF analysis. Inhibition of pump function as a result of cross-linking caused increased susceptibility to noxious compounds and reduction of N-phenylnaphthylamine efflux. Regain of function for impaired mutants was obtained upon exposure to the reducing agent DTT. The results support the presence of the asymmetric AcrB trimer in E. coli membranes and the functional rotation mechanism.

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Figure 1: Visualization of the engineered disulfide bridges.
Figure 2: SDS-PAGE and western blot analysis of single- and double-cysteine mutants of AcrB_cl.
Figure 3: Effect of DTT on N-phenylnaphthylamine (NPN) efflux by E. coli BW25113ΔacrB producing AcrB_cl mutants with disulfide links between the indicated subdomains.

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Acknowledgements

MALDI-TOF measurements were performed at the Functional Genomics Center Zurich (FGCZ). We thank P. Gehrig from the FGCZ for assistance with the MALDI-TOF instrument. This work was supported by grants of the TR-SFB Zürich-Konstanz (to K.D.), the ETH Zürich, EMDO Stiftung and the FK of the University of Zürich (to K.M.P.).

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  1. Markus A Seeger

    Present address: Current address: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.,

Authors and Affiliations

  1. Institute of Physiology and Zurich Centre for Integrative Human Physiology (ZIHP), University of Zurich, Winterthurerstrasse 190, Zürich, CH-8057, Switzerland

    Markus A Seeger, Thomas Eicher, Lorenz Brandstätter, François Verrey & Klaas M Pos

  2. Institute of Microbiology, Swiss Federal Institute of Technology (ETH), Wolfgang-Pauli-Strasse 10, Zürich, CH-8093, Switzerland

    Markus A Seeger & Christoph von Ballmoos

  3. Department of Biology, University of Konstanz, Universitätsstrasse 10, M647, Konstanz, D-78457, Germany

    Kay Diederichs

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Contributions

M.A.S., C.v.B. and K.M.P. designed the experiments; M.A.S., C.v.B, T.E. and L.B. performed the experiments; M.A.S., F.V., K.D. and K.M.P. analyzed the data; M.A.S., K.D. and K.M.P. wrote the article.

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Correspondence to Klaas M Pos.

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Seeger, M., von Ballmoos, C., Eicher, T. et al. Engineered disulfide bonds support the functional rotation mechanism of multidrug efflux pump AcrB. Nat Struct Mol Biol 15, 199–205 (2008). https://doi.org/10.1038/nsmb.1379

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