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Papers In Press, published online ahead of print October 23, 2003
Department of Cell Membrane Biology, Osaka University, Institute for Scientific and Industrial Research, Ibaraki, Osaka 567-0047
Corresponding Author: akihito{at}sanken.osaka-u.ac.jp
We previously reported the crystal structure of the major multidrug exporter AcrB in Escherichia coli (Murakami, S., et al. Nature 419, 587-593, 2002). The extra-membrane headpiece of the AcrB trimer contains a central pore composed of three a-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. Out of the 21 mutants, D99C to P119C, five, 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, that is, on the wall of the pore. These observations strongly indicate the important role of this pore in the drug transport process. A NEM 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 a-helical turns. Two mutants (V105C and Q112C), which showed the greatest loss of activity of all 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.
J. Biol. Chem, 10.1074/jbc.M308893200
Submitted on August 12, 2003
Revised on October 21, 2003
Accepted on October 23, 2003
Extra-membrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport
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