Determinants of Substrate Recognition by the Escherichia coli Multidrug Transporter MdfA Identified on Both Sides of the Membrane

Abstract

The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteineless MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (⌬˜H؉, inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.

Manipulating the drug/proton antiport stoichiometry of the secondary multidrug transporter MdfA

“…2B), further suggesting that the residue at position 354 is located at the Mdr recognition pocket of MdfA. The effect of mutating site 354 is not surprising: previous studies have shown that the loop between TM10 and -11, especially site 335 (20,21), and TM11 (20) are located at one face of the promiscuous substrate-binding pocket of MdfA. Changes in the pocket have usually modified the substrate recognition profile of MdfA.…”

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

“…Plasmids-Plasmids for overexpression of MdfA or various single cysteine mutants have been previously described (6,9). pT7-5/Para/mdfA 6HIS encoding the single cysteine mutant G410C was generated utilizing a standard PCR method with mutagenic oligonucleotide primers and a plasmid encoding Cys-less MdfA as a template.…”

“…Or do dissimilar substrates induce different conformational changes, all of which facilitate transport? Although it was shown that substrate binding indeed 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.…”

“…Binding-We have previously employed a genetic screen to study how MdfA interacts with its substrates (6). This assay identified residues that potentially participate in Mdr recognition.…”

Alkalitolerance: A biological function for a multidrug transporter in pH homeostasis

“…The substrate-recognition profile of MdfA includes lipophilic cations, zwitterionic drugs, and neutral compounds (10,11,19), and Glu-26 in transmembrane helix 1 of the transporter plays an important role in multidrug recognition (14,20,21). Transport experiments have shown that MdfA is driven by the proton electrochemical gradient and functions as a drug͞proton antiporter (11,22).…”