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J. Bacteriol. doi:10.1128/JB.01127-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Site-Directed Disulfide Cross-Linking Shows that Cleft Flexibility in the Periplasmic Domain is Needed for the Multidrug Efflux Pump AcrB of Escherichia coli

Department of Molecular and Cell Biology, University of California, Berkeley, California 94720-3202

^* To whom correspondence should be addressed. Email: nhiroshi{at}berkeley.edu.

	Abstract

The Escherichia coli AcrB is a multidrug efflux transporter that recognizes multiple toxic chemicals of diverse structures. Recent crystallographic studies of the asymmetric trimer of AcrB suggest that each protomer in the trimeric assembly goes through a cycle of conformational changes during drug export. However, biochemical evidence for this conformational change has not been provided. In this study, we took advantage of the observation that the external large cleft in the periplasmic domain of AcrB appears to become closed in the crystal structure of one of the three protomers, and carried out in vivo cross-linking studies between cysteine residues introduced by site-directed mutagenesis on both sides of the cleft, as well as at the interface between the periplasmic domain of AcrB trimer. Double cysteine mutants in the cleft or the interface were inactive. That this was due to the formation of disulfide bonds was suggested by the restoration of transport activity of the cleft mutants in a dsbA strain, which has diminished activity to form disulfide bonds in the periplasm. Furthermore, rapidly reacting, sulfhydryl-specific chemical cross-linkers, MTSs, inactivated the AcrB transporter with double cysteines in the cleft, expressed in dsbA cells, and this inactivation could be observed within a few seconds after the addition of cross-linker, in real time, by the increased ethidium influx into cells. These observations indicate that conformational changes including the closure of the external cleft in periplasmic domain is required for drug transport by AcrB.