In situ structure and assembly of the multidrug efflux pump AcrAB-TolC

Abstract

Multidrug efflux pumps actively expel a wide range of toxic substrates from the cell and play a major role in intrinsic and acquired drug resistance. In Gram-negative bacteria, these pumps form tripartite assemblies that span the cell envelope. However, the in situ structure and assembly mechanism of multidrug efflux pumps remain unknown. Here we report the in situ structure of the Escherichia coli AcrAB-TolC multidrug efflux pump obtained by electron cryo-tomography and subtomogram averaging. The fully assembled efflux pump is observed in a closed state under conditions of antibiotic challenge and in an open state in the presence of AcrB inhibitor. We also observe intermediate AcrAB complexes without TolC and discover that AcrA contacts the peptidoglycan layer of the periplasm. Our data point to a sequential assembly process in living bacteria, beginning with formation of the AcrAB subcomplex and suggest domains to target with efflux pump inhibitors.

Fig. 1

Visualizing the AcrAB–TolC efflux pump in the E. coli cell envelope. a A single slice from a tomogram of E. coli. The condensed materials shown inside of the cell are inclusion bodies resulting from membrane protein overexpression. b Zoomed inside view of the cell envelope containing the AcrAB–TolC pump which is indicated by the red rectangle. c Corresponding three-dimensional annotation of b showing the outer membrane (OM; blue), the inner membrane (IM; blue), peptidoglycan (PG; yellow), and the AcrAB–TolC pump (cyan). d Top view of the cell envelope containing the AcrAB–TolC pumps which appear as ring-shaped densities (indicated by the red circle)

Fig. 2

In situ Cryo-ET structures of the fully assembled AcrAB–TolC complex. a The side-view projection of the subtomogram average of the pump in presence of antibiotics. b Isosurface rendering of a fitted with high resolution cryo-EM model (PDB: 5V5S). c A slice through b showing a constriction at the boundary between AcrA and TolC (indicated by the red arrow). d A slice through the density map of the pump in presence of AcrB inhibitor (MBX3132). e Isosurface rendering of b overlaid with density map of the cell envelope

Fig. 3

In situ Cryo-ET structure of the AcrAB subcomplex. a The side-view projection of the subtomogram average of the AcrAB subcomplex in presence of antibiotics. b Isosurface rendering of a fitted with the cryo-EM single-particle model (PDB: 5V5S). c A slice through the density map of b. d Isosurface rendering of b overlaid with density map of the cell envelope

Fig. 4

Proposed in vivo assembly and functioning mechanism for multidrug efflux pump AcrAB–TolC. First, AcrB associates with AcrA to form the bipartite complex AcrAB. Next, AcrA changes its conformation to recruit TolC. Once TolC binds with the AcrAB bipartite complex, the fully assembled tripartite pump remains in the resting state. When AcrB encounters a drug molecule, the pump adopts an open conformation accompanied with a contraction along the long axis and the substrate is expelled through the channel and out of the cell