A periplasmic drug-binding site of the AcrB multidrug efflux pump: a crystallographic and site-directed mutagenesis study

Abstract

The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-beta-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.

FIG. 1.

The structure of the AcrB trimer cocrystallized with ciprofloxacin. The three protomers are shown in blue, mauve, and green, and the ciprofloxacin molecules are shown as CPK models. A. The wild-type AcrB trimer (from PDB file 1OYE). The locations of the “funnel,” “pore,” and “central cavity” (defined in reference 23) are shown in dotted lines. The entrances to the surface cavities, “depression,” and “fissure” (in the latter case, the right-hand entrance as viewed from the outside of the trimer) are shown in continuous lines. B. The N109A mutant AcrB trimer (this study). The figures were drawn with Accelrys Viewer Pro and POV-RAY 3.5.

FIG. 2.

Overlay of the backbones of N109A (unliganded) and wild-type AcrB (unliganded) proteins. The model of the unliganded N109A AcrB (this work) was overlaid on the model of unliganded wild-type AcrB determined by Murakami et al. (23), using the “magic fit” function of the DeepView program, and the N109A structure was colored in rainbow colors; red shows the largest deviation and blue the smallest deviation from the reference, wild-type AcrB structure. The A109 residue is shown as a red stick model (arrow). The reference structure is not shown. The deviation at the N-terminal end of TM8 is about 1 Å, which gradually increases to about 2.5 Å at the end of TM12. Although these values are small in comparison with the resolution of the structure, they appear to be significant, because many consecutive residues show a consistent pattern. The figure was created with DeepView and POV-RAY 3.5.

FIG. 3.

Simulated annealing omit maps of bound Cip (top) and R6G (bottom) molecules in the central cavity (left) and at the periplasmic binding pocket (right). The electron density omit map (contoured at 1.5 σ) was calculated with a starting temperature of 2,000°K and by excluding the bound ligand from the model.

FIG. 4.

The periplasmic drug-binding pocket with five ligands. This is a composite figure showing the locations of the ligands in the periplasmic domain. The view is from the outside into the center of the trimer, with the transmembrane domain at the bottom. The ligands shown in stick models are Cip (gray), Et (orange), R6G (red), Naf (yellow), and MC (blue). The portion of the protein constituting the binding pocket (discussed in the text) is in deep blue. This figure and the following three figures were drawn with PyMol (W. L. Delano, PyMol Graphic System [www.pymol.org]).

FIG. 5.

Interaction of Cip with amino acid side chains in the periplasmic binding pocket of the N109A mutant AcrB. Amino acid residues that are within 6 Å of any atoms of the ligand are shown.

FIG. 6.

The wall of the central cavity with five ligands. This is a composite figure showing the locations of the bound ligands in the central cavity. The view is from the center of the cavity toward the wall, with the periplasmic domain at the top. The ligands are shown in stick models in the same colors as in Fig. 3: thus, Cip (gray), Et (orange), R6G (red), Naf (yellow), and MC (blue).

FIG. 7.

Interaction of Cip with amino acid side chains in the central cavity of the N109A mutant AcrB. Amino acid residues within 6 Å of any atoms of Cip (in gray) are shown in green.

FIG. 8.

Western blot analysis of the expression of AcrA and the mutant AcrB proteins. Total protein extracts of E. coli HNCE1b (ΔacrA::cat ΔacrB::kan ΔacrD) harboring the different plasmid constructs based on plasmid pAcrAB were separated by SDS-polyacrylamide gel electrophoresis (7.5% gel) and probed with polyclonal anti-AcrA antibodies (lower panels) or with polyclonal anti-AcrB antibodies (upper panels). Panels A and B show different sets of mutants; for example, the left part of panel A shows mostly double mutants containing N109A.

FIG. 9.

Et accumulation by intact cells of E. coli HNCE1b expressing various versions of AcrB protein together with the wild-type AcrA (except cells containing pAcrB). Cells were grown in LB, induced with 0.1 mM of IPTG for 1 h, harvested, washed, and resuspended in phosphate buffer as described in Materials and Methods. Accumulation time course was monitored with a spectrofluorometer. Et was used at a final concentration of 5 μM. Panel A shows AcrB mutants containing the F666A mutation; Panel B shows those containing the F664A mutation.