Extramembrane central pore of multidrug exporter AcrB in Escherichia coli plays an important role in drug transport.
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 alpha-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 alpha-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.
Murakami
Satoshi
S
Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
Tamura
Norihisa
N
Saito
Asami
A
Hirata
Takahiro
T
Yamaguchi
Akihito
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D005033:Ethylmaleimide / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011726:Pyridinium Compounds / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D003545:Cysteine; D029968:Escherichia coli Proteins; D005033:Ethylmaleimide; D008565:Membrane Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011726:Pyridinium Compounds; C083496:AcrB protein, E coli; C025042:2-(dimethylaminostyryl)-1-ethylpyridinium