title: β-Lactam selectivity of multidrug transporters AcrB and AcrD resides in the proximal binding pocket.
abstract: β-Lactams are mainstream antibiotics that are indicated for the prophylaxis and treatment of bacterial infections. The AcrA-AcrD-TolC multidrug efflux system confers much stronger resistance on Escherichia coli to clinically relevant anionic β-lactam antibiotics than the homologous AcrA-AcrB-TolC system. Using an extensive combination of chimeric analysis and site-directed mutagenesis, we searched for residues that determine the difference in β-lactam specificity between AcrB and AcrD. We identified three crucial residues at the "proximal" (or access) substrate binding pocket. The simultaneous replacement of these residues in AcrB by those in AcrD (Q569R, I626R, and E673G) transferred the β-lactam specificity of AcrD to AcrB. Our findings indicate for the first time that the difference in β-lactam specificity between AcrB and AcrD relates to interactions of the antibiotic with residues in the proximal binding pocket.
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; Faculty of Pharmaceutical Science, Teikyo Heisei University, Nakano, Nakano-ku, Tokyo 164-8530, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Kobayashi
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Norihisa
authors.initials: N
authors.lastname: Tamura
authors.affiliation: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, United Kingdom.
authors.forename: Hendrik W
authors.initials: HW
authors.lastname: van Veen
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
authors.forename: Akihito
authors.initials: A
authors.lastname: Yamaguchi
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan; Department of Life Science, Tokyo Institute of Technology, Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Electronic address: murakami@bio.titech.ac.jp.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Murakami
mesh_terms: D000900:Anti-Bacterial Agents / Q000737:chemistry; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects; D018360:Crystallography, X-Ray; D024901:Drug Resistance, Multiple, Bacterial; D004926:Escherichia coli / Q000187:drug effects* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D021381:Protein Transport; D013379:Substrate Specificity; D047090:beta-Lactams / Q000737:chemistry*
chemical_list: D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D047090:beta-Lactams; C083496:AcrB protein, E coli; C000588785:AcrD protein, E coli
keywords: Antibiotic Resistance; Drug Resistance; Membrane Proteins; Membrane Transport; Multidrug Transporters