title: Role of Aromatic and Negatively Charged Residues of DrrB in Multisubstrate Specificity Conferred by the DrrAB System of Streptomyces peucetius.
abstract: Resistance to the anticancer antibiotics, doxorubicin and daunorubicin, in the producer organism Streptomyces peucetius is conferred by an ABC transporter made of two proteins, DrrA and DrrB, which together form a dedicated exporter for these two antibiotics. Surprisingly, however, the DrrAB system exhibits broad substrate specificity overlapping with well-studied multidrug resistance transporters, including P-glycoprotein. Therefore, it provides an excellent model for studying the molecular basis of multispecificity in a prototype efflux system with the potential to unravel the origin and evolution of multidrug resistance. It has been suggested that multispecificity in multidrug exporters may be generally determined by the number and location of aromatic residues. Strategically placed negatively charged residues may also be critical for binding of cationic lipophilic drugs. We selected 13 aromatic and four negatively charged residues on the basis of their location in and/or near the predicted drug-binding pocket of DrrB for analysis. Indeed, mutations of most tested residues drastically inhibited doxorubicin efflux. Interestingly, several mutants lost resistance to doxorubicin and verapamil simultaneously but retained resistance to Hoechst 33342 and/or ethidium bromide, suggesting the presence of overlapping as well as independent drug-binding sites in a common drug-binding pocket of DrrB. This study provides the first comprehensive analysis of residues involved in drug binding in a bacterial multidrug resistance protein of the ABC superfamily, and it shows strong similarity in the molecular mechanism of polyspecific drug recognition between DrrAB and Pgp. Altogether, we conclude that aromatic residue-based multidrug specificity is conserved across domains and over long evolutionary periods. The significance of these findings is discussed.
authors.affiliation: Department of Biology, Georgia State University , Atlanta, Georgia 30303, United States.
authors.forename: Kenneth
authors.initials: K
authors.lastname: Brown
authors.affiliation: Department of Biology, Georgia State University , Atlanta, Georgia 30303, United States.
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Biology, Georgia State University , Atlanta, Georgia 30303, United States.
authors.forename: Parjit
authors.identifier: 0000-0002-0864-3110
authors.initials: P
authors.lastname: Kaur
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism; D003630:Daunorubicin / Q000737:chemistry* / Q000378:metabolism; D004317:Doxorubicin / Q000737:chemistry* / Q000378:metabolism; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D004996:Ethidium / Q000737:chemistry / Q000378:metabolism; D019143:Evolution, Molecular; D015870:Gene Expression; D008958:Models, Molecular; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009154:Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D014700:Verapamil / Q000737:chemistry* / Q000378:metabolism
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D001562:Benzimidazoles; D003630:Daunorubicin; D004317:Doxorubicin; D004996:Ethidium; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D011994:Recombinant Proteins; D014700:Verapamil; C104038:DrrB protein, Streptomyces peucetius; C017807:bisbenzimide ethoxide trihydrochloride