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Journal of Bacteriology, October 2006, p. 7290-7296, Vol. 188, No. 20
0021-9193/06/$08.00+0     doi:10.1128/JB.00684-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Conformation of the AcrB Multidrug Efflux Pump in Mutants of the Putative Proton Relay Pathway

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa 50011,¹ Department of Physics and Astronomy, Iowa State University, Ames, Iowa 50011,² Department of Molecular and Cell Biology, University of California, Berkeley, California 94720,³ Berkeley Center for Structural Biology, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720⁴

Received 12 May 2006/ Accepted 25 July 2006

We previously reported the X-ray structures of wild-type Escherichia coli AcrB, a proton motive force-dependent multidrug efflux pump, and its N109A mutant. These structures presumably reflect the resting state of AcrB, which can bind drugs. After ligand binding, a proton may bind to an acidic residue(s) in the transmembrane domain, i.e., Asp407 or Asp408, within the putative network of electrostatically interacting residues, which also include Lys940 and Thr978, and this may initiate a series of conformational changes that result in drug expulsion. Herein we report the X-ray structures of four AcrB mutants, the D407A, D408A, K940A, and T978A mutants, in which the structure of this tight electrostatic network is expected to become disrupted. These mutant proteins revealed remarkably similar conformations, which show striking differences from the previously known conformations of the wild-type protein. For example, the loop containing Phe386 and Phe388, which play a major role in the initial binding of substrates in the central cavity, becomes prominently extended into the center of the cavity, such that binding of large substrate molecules may become difficult. We believe that this new conformation may mimic, at least partially, one of the transient conformations of the transporter during the transport cycle.

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