Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system

Mol Microbiol. 2004 Jul;53(2):697-706. doi: 10.1111/j.1365-2958.2004.04158.x.


The major Escherichia coli multidrug efflux pump AcrAB-TolC expels a wide range of antibacterial agents. Using in vivo cross-linking, we show for the first time that the antiporter AcrB and the adaptor AcrA, which form a translocase in the inner membrane, interact with the outer membrane TolC exit duct to form a contiguous proteinaceous complex spanning the bacterial cell envelope. Assembly of the pump appeared to be constitutive, occurring in the presence and absence of drug efflux substrate. This contrasts with substrate-induced assembly of the closely related TolC-dependent protein export machinery, possibly reflecting different assembly dynamics and degrees of substrate responsiveness in the two systems. TolC could be cross-linked independently to AcrB, showing that their large periplasmic domains are in close proximity. However, isothermal titration calorimetry detected no interaction between the purified AcrB and TolC proteins, suggesting that the adaptor protein is required for their stable association in vivo. Confirming this view, AcrA could be cross-linked independently to AcrB and TolC in vivo, and calorimetry demonstrated energetically favourable interactions of AcrA with both AcrB and TolC proteins. AcrB was bound by a polypeptide spanning the C-terminal half of AcrA, but binding to TolC required interaction of N- and C-terminal polypeptides spanning the lipoyl-like domains predicted to present the intervening coiled-coil to the periplasmic coils of TolC. These in vivo and in vitro analyses establish the central role of the AcrA adaptor in drug-independent assembly of the tripartite drug efflux pump, specifically in coupling the inner membrane transporter and the outer membrane exit duct.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Outer Membrane Proteins / metabolism*
  • Bacterial Outer Membrane Proteins / physiology
  • Biological Transport, Active
  • Carrier Proteins / metabolism*
  • Drug Resistance, Multiple, Bacterial*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / metabolism*
  • Gene Expression Regulation, Bacterial
  • Lipoproteins / metabolism*
  • Membrane Proteins / metabolism*
  • Membrane Transport Proteins / metabolism
  • Membrane Transport Proteins / physiology
  • Multidrug Resistance-Associated Proteins
  • Protein Binding
  • Protein Interaction Mapping
  • Protein Structure, Tertiary


  • AcrA protein, E coli
  • AcrB protein, E coli
  • Bacterial Outer Membrane Proteins
  • Carrier Proteins
  • Escherichia coli Proteins
  • Lipoproteins
  • Membrane Proteins
  • Membrane Transport Proteins
  • Multidrug Resistance-Associated Proteins
  • tolC protein, E coli