A single membrane-embedded negative charge is critical for recognizing positively charged drugs by the Escherichia coli multidrug resistance protein MdfA

The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia co li multidrug transporter MdfA, the hydrophobicity profile and PhoA fusion a nalysis have so far identified only one membrane-embedded charged amino aci d residue (E26), In order to determine whether this negatively charged resi due may play a role in multidrug recognition, we evaluated the expression a nd function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance a ctivity against positively charged drugs, but retained chloramphenicol effl ux and resistance, In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and tran sport were drastically inhibited; however, the mutant exhibited almost wild -type multidrug resistance activity against lipophilic cations, These resul ts suggest that although the negative charge at position 26 is not essentia l for active transport, it dictates the multidrug resistance character of M dfA, We show that such a negative charge is also found in other drug resist ance transporters, and its possible significance regarding multidrug resist ance is discussed.