| Abstract
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Multidrug resistance plays a crucial role in the failure of drug-based treatment of cancer and various infectious diseases. One of the major resistance mechanisms is active export of different chemically unrelated drugs from cells by multidrug (Mdr) transporters. We use MdfA, a secondary Mdr transporter of the Major Facilitator Superfamily (MFS) from E. coli, as a model in our studies. MdfA is a 410-residue-long membrane protein with 12 transmembrane (TM) helices, which uses the proton electrochemical membrane potential to drive efflux of a variety of toxic compounds, including lipophilic, neutral, cationic, and zwitterionic ones. To better understand the mechanism of substrate recognition and translocation by secondary Mdr transporters, we investigated central structural and functional properties of MdfA. Initially, we studied the biochemical properties and oligomeric structure of the purified transporter in detergent solution. Purified MdfA was analyzed for its associated detergent (DDM) and phospholipids, its molecular weight, and substrate-binding activity. The results showed that MdfA is a functional monomer in DDM solution. This finding is crucial for studying various functional aspects of MdfA. Another important prerequisite for further investigation of MdfA is detailed structural information, which is so far unavailable in the form of high-resolution x-ray model. Assuming that MdfA may have helix packing organization that is similar to those of other MFS members with known tertiary structure, we generated a 3D model for MdfA using a combination of several approaches: kPROT, correlated mutations, and structural alignment. The resulted model generally supports previous structural predictions, regarding the secondary structure and membrane topology of MdfA and also the notion that residue Glu26 is located inside TM1. Interestingly however, according to the 3D model, MdfA may have two additional membrane-embedded charged residues, Asp34 and Arg112. Further characterization of these residues demonstrated that whereas Asp34 comprises, together with Glu26, an essential pair of acidic residues (see later), studies of Arg112 showed that a positive charge at this position is absolutely essential for MdfA function. To approach the mechanism of proton recognition and translocation by MdfA, we investigated the role of all of its negatively charged residues. Interestingly, these studies revealed that no single acidic residue plays an irreplaceable role in the transport activity of MdfA. Nevertheless, we obtained evidence in support of the notion that also MdfA utilizes an essential membrane embedded negative charge for active transport. Surprisingly however, we showed that MdfA tolerates displacements of the essential negative charge to various locations in its putative drug translocation pathway (26, 34, 150). Such permissibility has never been described for substrate-specific secondary transporters, thus further illustrating the exceptional structural promiscuity of Mdr transporters. To summarize, this work contributed to better understanding of basic structural properties of secondary multidrug transporter MdfA from E. coli, revealed new insights to transport mechanism of MdfA and the their implications to the secondary multidrug transport.
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