Abstract
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 this protein were individually replaced by cysteine except for the initial methionine. As a result, we could obtain a complete map of the functionally or structurally important residues. In addition, we could determine the precise boundaries of all the transmembrane segments on the basis of the reactivity with N-ethylmaleimide (NEM). The NEM binding results indicated the presence of a transmembrane water-filled channel in the transporter. The twelve transmembrane segments can be divided into three groups; four are totally embedded in the hydrophobic interior, four face a putative water-filled channel along their full length, and the remaining four face the channel for half their length, the other halves being embedded in the hydrophobic interior. These three types of transmembrane segments are mutually arranged with a 4-fold symmetry. The competitive binding of membrane-permeable and -impermeable SH reagents in intact cells indicates that the transmembrane water-filled channel has a thin barrier against hydrophilic molecules in the middle of the transmembrane region. Inhibition and stimulation of NEM binding in the presence of tetracycline reflects the substrate-induced protection or conformational change of the Tn10-encoded metal-tetracycline/H(+) antiporter. The mutations protected from NEM binding by tetracycline were mainly located around the permeability barrier in the N-terminal half, suggesting the location of the substrate binding site.
Highlights
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
We previously reported that putative transmembrane helices (TM) 3 [6], 6 [7], and 9 [8] are totally embedded in a highly hydrophobic environment, because none of the cysteine-scanning mutants as to these transmembrane helices reacted with a maleimide derivative, N-ethylmaleimide (NEM), whereas cysteine mutants as to putative loop regions are generally highly reactive with NEM, except for those as to a small number of non-reactive positions [9]
The Effect of Tetracycline on NEM Binding—First of all, we investigated the effect of tetracycline on the [14C]NEM binding to 76 NEM-reactive cysteine-scanning mutants as to loop6 –7, TM7, loop7– 8, TM10, TM12, the C terminus, and their flanking regions
Summary
TetA(B), Tn10-encoded metal-tetracycline/Hϩ antiporter; TM, transmembrane helices; NEM, N-ethylmaleimide; AMS, 4-acetamido-4Ј-maleimidylstilbene-2,2Ј-disulfonic acid; proteins. It belongs to a major facilitator superfamily [3], and its 12-membrane-spanning structure (Fig. 1) was established by site-directed competitive chemical modification of cysteine mutants of a cysteine-free TetA(B) [5]. We have obtained a complete set of the 400 cysteine-scanning mutants of TetA(B), except for the mutant as to the initial methionine. In this manuscript, we report the results of NEM binding and competitive binding of NEM and a membrane-impermeable sulfhydryl reagent, 4-acetamido-4Јmaleimidylstilbene-2,2Ј-disulfonic acid (AMS), in comparison. We first drew a complete map of the wall of the transmembrane water-filled channel and demonstrated the presence of a permeability barrier for hydrophilic molecules in the middle of the channel
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