Abstract
Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.
Highlights
Mdr transporters exist in many families of transport proteins that utilize various transport mechanisms [1, 2]
We studied this question by utilizing several approaches for detecting conformational changes in the Escherichia coli Mdr transporter MdfA, which serves as a model of secondary Mdr transport [7]
To investigate substrate binding further, we labeled the putative pocket by a fluorescence probe covalently bound at position 147, utilizing a functional Cys-less mutant with a single cysteine inserted at this position (MdfA-A147C)
Summary
Mdr transporters exist in many families of transport proteins that utilize various transport mechanisms [1, 2]. The research of recent years has shed light on the major biochemical properties of Mdr transporters These transporters contain large substrate-binding pockets and can extract their substrates from the cytoplasm and/or the membrane. It was shown that substrate binding induces conformational changes in Mdr transporters [5, 6], it is not understood how a single transporter can be “conformationally responsive” to the binding of a diverse group of compounds, such that all of them induce structural rearrangements in the protein that facilitate transport. We conclude that MdfA has a sensitive conformational switch that can be triggered either by substrate binding or by attaching unrelated agents inside the pocket
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