Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer

Abstract

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked efflux. We have determined the three-dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A resolution. The structure of EmrE consists of a bundle of eight transmembrane alpha-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.

Fig. 1. Lattice line data. Plots of amplitudes (lower panels) and phases (upper panels) along the z* axis for three selected reflections. The fitted lattice lines were produced by weighted least squares fitting and the resulting errors are shown.

Fig. 2. Top and side view of the 3D density map of EmrE. (A) Top view perpendicular to the membrane plane of the density contoured at 1.2 σ. (B) Schematic view perpendicular to the membrane plane of the architecture of EmrE with all helices (A–H) approximated as straight cylinders. (C) Side view along the membrane plane of the density contoured at 1.2 σ. (D) Side view of a slice along the membrane plane of the density contoured at 0.8 σ, to emphasize the location of the substrate TPP⁺. The eight idealized helices were placed manually into the map and were not subjected to refinement. The helices are grouped into two sets: those coloured yellow form the substrate binding pocket and those coloured red are separated from the binding pocket by helices C and F. The maps were analysed and the idealized helices generated in the environment of O (Jones et al., 1991).

Fig. 3. Horizontal slices through the density map: 3Å-deep slices through the density map (contoured at 0.8 σ) perpendicular to the membrane plane separated by 5 Å. The panels show sections above (A and B) and below (D and E) the centre (C). The six yellow helices form the wall of the substrate binding pocket, whereas the two red helices are separated from the binding pocket. The density at the centre of the yellow helices in sections A–C is believed to represent the substrate TPP⁺.

Fig. 4. Packing of EmrE dimers in the 2D crystals. The two different tetrameric arrangements of EmrE dimers as observed in the p2 crystals are shown with all helices approximated as straight cylinders. Each dimer is numbered and dimer 1 is identical to the one shown in Figure 2B. The 2-fold axes perpendicular and parallel to the membrane plane are shown in white.

Fig. 5. Possible arrangements of monomers in the dimer. (A) Four possible monomer boundaries in the EmrE dimer are shown. The transmembrane helices are depicted as circles distributed as in the central section of the structure shown in Figure 3C. A red circle depicts the location of the substrate TPP⁺. A continuous green line illustrates the hypothetical interface between monomers. (B) View of the density map contoured at 1.0 σ to show the connection of density between helix H and helix F (white arrow). The density for TPP⁺ is also shown for reference (dashed arrow). The view is along helix G and from the same side as in Figure 2.

Fig. 6. Transport mechanism in EmrE. (A) Cartoon of EmrE based upon the 3D structural analysis, with TPP⁺ (depicted by a red sphere) bound in the centre of a cavity open to the cytoplasm and one leaflet of the lipid bilayer. (B) Schematic indicating the minimal structural changes during electrogenic TPP⁺ efflux suggested by the 3D structure. EmrE binds TPP⁺ from either the inner leaflet of the membrane or from the cytoplasm (1) and a conformational change (*) involving the change in tilt of helix H relative to helix A occurs to open the binding chamber to the periplasm (2). Proton binding displaces TPP⁺ into the periplasm (3), and a second proton-driven conformation change (*) re-orients the binding site to face the cytoplasm (4). TPP⁺ is represented as a red ball and protons as blue balls, labelled ‘+’.