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J. Biol. Chem., Vol. 279, Issue 5, 3743-3748, January 30, 2004

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Extramembrane Central Pore of Multidrug Exporter AcrB in Escherichia coli Plays an Important Role in Drug Transport^*

Satoshi Murakami¶||, Norihisa Tamura, Asami Saito, Takahiro Hirata¶, and Akihito Yamaguchi¶**

From the Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan, Faculty of Pharmaceutical Science, Osaka University, Suita, Osaka 565-0871, Japan, ^¶CREST, Japan Science and Technology Corporation, Osaka 567-0047, Japan, and ^||PRESTO, Japan Science and Technology Corporation, Osaka 567-0047, Japan

We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., Nakashima, R., Yamashita, E., and Yamaguchi, A. (2002) Nature 419, 587-593). The extramembrane headpiece of the AcrB trimer contains a central pore composed of three -helices. Each pore helix belongs to a different monomer. In this study, we constructed cysteine-scanning mutants as to the residues comprising the pore helix. Of the 21 mutants (D99C to P119C), 5 (D101C, V105C, N109C, Q112C, and P116C) showed significantly reduced drug resistance and drug-exporting activity. These residues are localized on one side of the pore helix, i.e. on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A N-ethylmaleimide binding experiment revealed that the pore is in the closed state, and the thickness of the permeability barrier in the middle of the pore corresponds to 2.5 -helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all of the pore mutants, were detected in the form of disulfide cross-linking dimers under normal conditions, suggesting that a conformational change of the pore is indispensable during the transport process.

Received for publication, August 12, 2003 , and in revised form, October 21, 2003.

The atomic coordinates (code 1IWG) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants-in-aid from the Ministry of Education and the Ministry of Science and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed: Institute of Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki-shi, Osaka 567-0047, Japan. Tel.: 81-6-6879-8545; Fax: 81-6-6879-8549; E-mail: akihito{at}sanken.osaka-u.ac.jp.

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