Complete cysteine-scanning mutagenesis and site-directed chemical modification of the Tn10-encoded metal-tetracycline/H+ antiporter

Bacterial Tn10-encoded metal-tetracycline/H+ antiporter was the first found drug exporter and has been studied as a paradigm of antiporter-type major facilitator superfamily transporters. Here the 400 amino acid residues of t his protein were individually replaced by cysteine except for the initial m ethionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the pre cise boundaries of all the transmembrane segments on the basis of the react ivity with N-ethylmaleimide (NEM). The NEM binding results indicated the pr esence of a transmembrane water-filled channel in the transporter. The twel ve transmembrane segments can be divided into three groups; four are totall y embedded in the hydrophobic interior, four face a putative water-filled c hannel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic inte rior. These three types of transmembrane segments are mutually arranged wit h a 4-fold symmetry. The competitive binding of membrane-permeable and -imp ermeable SH reagents in intact cells indicates that the transmembrane water -filled channel has a thin barrier against hydrophilic molecules in the mid dle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection o r conformational change of the Tn10-encoded metal-tetracycline/H+ antiporte r, The mutations protected from NEM binding by tetracycline were mainly loc ated around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.