Structure of the tripartite multidrug efflux pump AcrAB-TolC suggests an alternative assembly mode

Abstract

Escherichia coli AcrAB-TolC is a multidrug efflux pump that expels a wide range of toxic substrates. The dynamic nature of the binding or low affinity between the components has impeded elucidation of how the three components assemble in the functional state. Here, we created fusion proteins composed of AcrB, a transmembrane linker, and two copies of AcrA. The fusion protein exhibited acridine pumping activity, suggesting that the protein reflects the functional structure in vivo. To discern the assembling mode with TolC, the AcrBA fusion protein was incubated with TolC or a chimeric protein containing the TolC aperture tip region. Three-dimensional structures of the complex proteins were determined through transmission electron microscopy. The overall structure exemplifies the adaptor bridging model, wherein the funnel-like AcrA hexamer forms an intermeshing cogwheel interaction with the α-barrel tip region of TolC, and a direct interaction between AcrB and TolC is not allowed. These observations provide a structural blueprint for understanding multidrug resistance in pathogenic Gram-negative bacteria.

Keywords: complex structure; electron microscopy; membrane protein; multidrug efflux pump.

Fig. 1.

Construction of the AcrBA fusion proteins and acridine pumping activities. (A) Schematic representations of the construction of the AcrBA fusion protein. (B) Acridine pumping activities of the AcrBA fusion proteins. Each E. coli strain BW25113(DE3) ΔacrAB transformed with pET22b-AcrB-TM#(1-6)-AcrA-AcrA was cultured on an acridine-containing LB agar plate, and the pumping activities of the fusion proteins were measured. The E. coli strain with the pET22b vector containing the wild type AcrA and AcrB genes (AcrA, AcrB) as well as the empty pET22b vector (2) were used as controls.

Fig. 2.

TEM images and a docked model of the AcrAB-TolC efflux pump. (A) A raw electron micrograph of the purified AcrBA fusion protein and TolC complex as well as representative class averages (inset). (B) 3D reconstruction of the complex. (C) Docked pseudoatomic model of the ternary complex that encompasses the inner membrane (IM), outer membrane (OM), and periplasmic space. (D) Docking the “adaptor wrapping model” into the 3D reconstruction and the section along the symmetry axis. AcrB is colored blue, AcrA is in yellow, and TolC is in red. The scale bars are 100 nm and 10 nm in (A) and the inset, respectively.

Fig. 3.

TEM images and a docked model of the AcrBA fusion protein as well as the TolC α-hairpin tip-containing MacA protein. (A) A raw electron micrograph of the purified AcrBA fusion protein and MacA-TolCα-hybrid dimer complex as well as representative class averages (inset). (B) 3D reconstruction of the complex and (C) the docked pseudoatomic model. Sections along and perpendicular to the symmetry axis are shown. AcrB is colored blue, AcrA is in yellow, the TolC α-hairpin tip is in and MacA is in green. The dilated binding interface between AcrA and the TolC α-hairpin tip is indicated by an arrow. The scale bars are 100 nm and 10 nm in (A) and the inset, respectively.

Fig. 4.

Intermeshing cogwheel interactions between the AcrA hexamer and TolC trimer in the open state. (A) The surface representative model (left) and its section along the symmetry axis (right) of the binding interface between the AcrA hexamer and TolC trimer based on the intermeshing cogwheel interactions. (B) The complex model and the section of the AcrA hexamer and TolC trimer, which is docked into 3D reconstructions of the AcrBA fusion protein and TolC (AcrBA-TolC) described in Fig. 2. (C) The complex model is docked into the cryo-EM density map reported in Du et al. (2014) (AcrBA-AcrAZ-TolC; EMDB ID: EMD-5915) The EM maps are shown in surface, the AcrA hexamer is colored yellow, and TolC is in red.