RESUMO
The mechanism by which multidrug transporters interact with structurally unrelated substrates remains enigmatic. Based on transport competition experiments, photoaffinity labeling, and effects on enzymatic activities, it was proposed in the past that multidrug transporters can interact simultaneously with a number of dissimilar substrate molecules. To study this phenomenon, we applied a direct binding approach and transport assays using the Escherichia coli multidrug transporter MdfA, which exports both positively charged (e.g., tetraphenylphosphonium, TPP(+)), zwitterionic (e.g., ciprofloxacin), and neutral (e.g., chloramphenicol) drugs. The interaction of MdfA with various substrates was examined by direct binding assays with the purified transporter. The immobilized MdfA binds TPP(+) in a specific manner, and all the tested positively charged substrates inhibit TPP(+) binding. Surprisingly, although TPP(+) binding is not affected by zwitterionic substrates, the neutral substrate chloramphenicol stimulates TPP(+) binding by enhancing its affinity to MdfA. In contrast, transport competition assays show inhibition of TPP(+) transport by chloramphenicol. We suggest that MdfA binds TPP(+) and chloramphenicol simultaneously to distinct but interacting binding sites, and the interaction between these two substrates during transport is discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Compostos de Benzalcônio/farmacologia , Benzimidazóis/farmacologia , Transporte Biológico/efeitos dos fármacos , Cloranfenicol/metabolismo , Cloranfenicol/farmacologia , Daunorrubicina/farmacologia , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli/genética , Etídio/farmacologia , Oniocompostos/antagonistas & inibidores , Oniocompostos/metabolismo , Compostos Organofosforados/antagonistas & inibidores , Compostos Organofosforados/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Solubilidade , Trítio/metabolismoRESUMO
The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each can recognize and expel a broad spectrum of chemically unrelated drugs from the cell. Despite extensive research for many years, the actual mechanism of multidrug transport is still largely unknown. In addition to general questions dealing with energy coupling, the molecular view of substrate recognition by Mdrs is generally obscure. This mini-review describes structural and functional properties of the Escherichia coli Mdr, MdfA, and discusses the possibility that this transporter may serve as a model for studying the multidrug recognition phenomenon and the mechanism of multidrug transport.
