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From Proteopedia

The E. coli AcrB Efflux Pump

The is part of a tripartite system used by E. Coli to remove antibiotic and other toxic molecules from the bacterial cell. It works in tandem with , an outer membrane pore can be recruited for many purposes. The beta barrel at the top of the TolC protein spans the outer membrane, allowing the drug to exit the bacterium.

Structural Elements

The AcrB pump is a homotrimer with monomers of 1049 amino acids. The protein has three domains: a transmembrane domain that spans the inner membrane of the bacterium and is responsible for energy transduction, a porter domain which is responsible for selectivity and transport of substrate, and a TolC docking domain that funnels exported substrate into the TolC channel protein. These domains are made up of : alpha helices in the TM region and a mixture of alpha helices and beta sheets in the other two domains. There is a lot of "random coil" (shown in blue) inside the porter domain of each monomer. Many of the residues responsible for binding and moving substrate are found in these primary structures.

A monomer colored red to blue from shows that the three domains are not assembled according to the order of their primary structure.

Substrates

Because of the large binding pockets in AcrB, it is able to bind and export a wide variety of structurally dissimilar antibiotic drugs such as ethidium, ciprofloxacin, rifampicin, erythromycin, oxacillin, minocycline, acridine, and rhodamine 6G (Pos 2009). The reason for this wide range of substrates is the presence of multiple different entry and binding sites within the AcrB porter domain. Different drugs will bind to different residues in the binding sites depending on their size and polarity. Recent experiments (Nakashima, et al. 2011; Eicher, et al. 2012) have found two main binding sites separated by a , which contains three Phe residues (pink). The position of this loop changes in the transition from L to T: in the L conformer, the switch loop occludes some larger drugs from the distal binding pocket. In the T conformer, the switch loop blocks the exit of the drug back into the periplasm.

Minocycline is a lower molecular weight drug (~400 g/mol) and was one of the first to be cocrystallized with AcrB. It binds in the in the T monomer (residues within 4 Angstroms of the drug shown in pink). It is unclear whether the drug skips the proximal binding pocket entirely and goes straight to the distal pocket while the monomer is in the L state, or goes from proximal to distal with the L--T conformation. Here, the can be seen (in green) in front of minocycline, trapping it in the distal binding pocket. (blue=polar, green=aliphatic, orange=hydrophobic) also hold the drug in the binding pocket.

Rifampicin is a high molecular weight drug (~800 g/mol) that has been crystallized in the (interacting residues highlighted in teal and shown in ball-and-stick representation). The proximal pocket was only discovered upon cocrystallization of AcrB with large drugs, such as rifampicin and erythromycin. In this case, the switch loop is located (switch loop is shown in orange). interacting residues are shown here (Polar in blue, nonpolar in orange).

(Rifampicin crystal structure from Nakashima, et al. 2011)

Energy Transduction

There are (Asp408, Asp407, Lys940, and Arg 971) whose protonation and deprotonation are suggested to play a large role in the conformational change between the L, T, and O states (Eicher, et al. 2009). This mechanism has been likened to the proton translocation-driven functional rotation of the monomers in ATP synthase (Pos, 2009).

References

Eicher, Thomas, et al. 2012. Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proceedings of the National Academy of Sciences 109: 5687-5692.

Nakashima, Ryosuke, Keisuke Sakurai, Seiji Yamasaki, Kunihiko Nishino, and Akihito Yamaguchi. 2011. Structures of the multidrug exporter AcrB reveal a proximal multisite drug-binding pocket. Nature 480: 565-570.

Pos, Klaas M. 2009. Drug transport mechanism of the AcrB efflux pump. Biochimica et Biophysica Acta 1794: 782-793.