Figure 5.

Structure of asymmetric AcrB comprising three protomers in the loose (L, blue), tight (T, yellow), and open (O, red) conformation. (A) Side view of trimeric AcrB along the membrane plane. Indicated are the transmembrane, periplasmic porter, and funnel domains (TMD, PD, and FD). (B) Top view on the porter domain. In the L protomer (blue), the PN1, PN2, PC1, and PC2 subdomains are indicated. The PC1 and PC2 subdomains constitute a cleft as part of the access pocket (AP, dashed oval). The T protomer contains also an open PC1/PC2 cleft but is less voluminous compared to the one in the L protomer. Between the PN2 and PC1 subdomains, the deep binding pocket (DBP, dashed oval) is indicated. The switch loop (green) separates the AP and the DBP. In the O protomer (red), the PC1/PC2 cleft, the AP, and the DBP are closed. From the closed DBP, a tunnel is present which exits at the funnel domain. In the asymmetric LTO AcrB porter domain representations (top view) on the left, binding of drugs to the access pocket is shown. Rifampicin (sphere representation in pink) binds in the L protomer (highlighted in blue) at the proximal side of the access pocket, and erythromycin (sphere representation in pink) binds to most distal part of the access pocket, underneath the switch loop (green).²⁸⁹ On the right, the T protomer is highlighted (in yellow), and bound minocycline (sphere representation in pink) is located in the distal groove region of the deep binding pocket,^288,290 whereas puromycin (sphere representation in cyan) binds to the more proximal cave region of the deep binding pocket underneath the switch loop (in green).³⁰⁵ (C) Top view of the funnel domain (FD). The N-terminal FN and the C-terminal FC subdomains are indicated. The AcrB trimer is stabilized by a loop protruding from the FN subdomain connected to the FD subdomain of the neighboring protomer emerging from subdomain DN to DC of the adjacent protomer. The distal FD remains largely unaffected by the conformational cycling and remains structurally unchanged during the LTO cycle. (D) Top view (from the periplasm) of the transmembrane domain (TMD). The TMD displays 12 transmembrane helices (TMHs). The proton relay network consists of residues D407, D408, and K940 associated with R971 and T978 located in the center of the TM-domains. Adjacent protomers interact via TM1 and TM8. The large central cavity is depicted by a circle. This image was constructed with PDB 4DX5 (in complex with minocycline)²⁹⁰ for the structure images in complex with drugs. Coordinates for rifampicin (PDB 3AOB), erythromycin (PDB 3AOC), minocycline (PDB 4DX5), and puromycin (PDB 5NC5) were superposed to the 4DX5 structure.