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. 2010 Apr 13;107(15):6559-65.
doi: 10.1073/pnas.1001460107. Epub 2010 Mar 8.

Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli

Yumiko Takatsuka  1 , Cheng Chen, Hiroshi Nikaido
Affiliations

Mechanism of recognition of compounds of diverse structures by the multidrug efflux pump AcrB of Escherichia coli

Yumiko Takatsuka et al. Proc Natl Acad Sci U S A. .

Abstract

The AcrB trimeric multidrug efflux transporter of Escherichia coli pumps out a very wide spectrum of compounds. Although minocycline and doxorubicin have been cocrystallized within the large binding pocket in the periplasmic domain of the binding protomer, nothing is known about the binding of many other ligands to this protein. We used computer docking to evaluate the interaction of about 30 compounds with the binding protomer and found that many of them are predicted to bind to a narrow groove at one end of the pocket whereas some others prefer to bind to a wide cave at the other end. Competition assays using nitrocefin efflux and covalent labeling of Phe615Cys mutant AcrB with fluorescein-5-maleimide showed that presumed groove-binders competed against each other, but cave-binders did not compete against groove-binders, although the number of compounds tested was limited. These results give us at least a hypothesis to be tested by more biochemical and genetic experiments in the future.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Some known substrates of the AcrB efflux pump.
Fig. 2.
Fig. 2.
An overview of the substrate-binding pocket in the Binding protomer of AcrB. (A) The side view of the asymmetric trimer of AcrB (21), with the Access protomer removed, so that the putative substrate tunnels (detected by using the program Caver (41) and shown in white) in the Binding protomer (in the bronze color) can be seen. The three possible points of entry for substrates are shown: CL (periplasmic cleft), V (vestibule between protomers, close to the membrane surface), and CA (a large internal cavity in the transmembrane domain). The Extrusion protomer is shown in green. The porter (P) and the TolC-docking subdomains (D) of the periplasmic domain, as well as the transmembrane domain (T) are also identified. (B) The view similar to A, but the proximal portion clipped away, to reveal the binding pocket composed of F136, V139, F178, I277, A279, E280, P285, Y327, F610, V612, F615, F617, I626, and F628, shown as surface with carbons in orange. The minocycline molecule cocrystallized in PDB file 2DRD (21) is shown as green sticks. (Inset) The enlargement of the binding pocket, in a view similar to Figs. 3 and 4 and Figs. S1 and S2. (C Inset) A top view, in which the proximal part is clipped away. The Access protomer is shown in mauve. The binding pocket, shown enlarged in C, is a narrow extension of the substrate tunnel, as shown. A part of minocycline is in the pocket, whereas the hydrophilic portion protrudes into the large tunnel.
Fig. 3.
Fig. 3.
Top five binding modes of minocycline (Top), nitrocefin (Middle), and fluorescein maleimide (Bottom) for the Binding protomer of AcrB (2DRD) predicted by Autodock Vina. The view in this figure, as well as in Fig. 4 and Figs. S1 and S2, is the side view of the binding pocket (shown as a surface with carbons in orange), similar to Fig. 2B Inset. It is from the outside of the Access protomer, which was removed by clipping together with much of the Extrusion and Binding protomers. The computer-predicted poses of the ligands are shown in stick models in CPK colors, and the minocycline in the crystal structure (2DRD) is shown for reference in a stick model with carbons in green. The poses are arranged in the order of calculated binding energy, beginning with the one predicted to be most stable at extreme left.
Fig. 4.
Fig. 4.
Some groove binders and cave binders predicted by Autodock Vina for the Binding protomer of AcrB. The numbers in parentheses indicate whether the pose shown is for the most favored one (1) or the second most favored one (2). The latter pose was chosen because the most favored pose showed binding outside the binding pocket (Fig. S2A).
Fig. 5.
Fig. 5.
Competition by minocycline and chloramphenicol for AcrB-catalyzed efflux of nitrocefin. Cells were incubated with varying external concentrations of nitrocefin in the presence of a potential competitor (0 or 50 μM), and the efflux rate was calculated as described in (28). Open symbols, no competitor; filled in symbols, with competitor.
Fig. 6.
Fig. 6.
Competition for fluorescein maleimide labeling of Phe615Cys mutant AcrB Binding protomer by some substrates of the pump. (A Left) composition of each construct tested. (Right) The extent of labeling was tested with 40 μM fluorescein maleimide and without competitors on all protomer units of the covalently linked AcrB trimer, in which the first protomer was forced to take the Extrusion conformation by the introduction of Asp407Asn mutation. (B) Potential competitors were added at different concentrations, and the extent of fluorescein labeling of the mutant AcrB was assessed as described in the Experimental Procedures, by using 20 μM fluorescein-5-maleimide.
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