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Journal of Bacteriology, October 2005, p. 6804-6815, Vol. 187, No. 19
0021-9193/05/$08.00+0     doi:10.1128/JB.187.19.6804-6815.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

Edward W. Yu,^1,2, Julio R. Aires,^1,, Gerry McDermott,³ and Hiroshi Nikaido^1*

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720,¹ Department of Physics and Astronomy, Iowa State University, Ames, Iowa 50011,² Berkeley Center for Structural Biology, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720³

Received 29 April 2005/ Accepted 7 July 2005

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-ß-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.

E.W.Y. and J.R.A. contributed equally to this study.

Present address: Faculté des Sciences Pharmaceutiques et Biologiques, Laboratoire de Microbiologie, 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France.

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