Cysteine-scanning Mutagenesis of Transmembrane Segments 4 and 5 of the Tn10-encoded Metal-Tetracycline/H+ Antiporter Reveals a Permeability Barrier in the Middle of a Transmembrane Water-filled Channel

Abstract

Cysteine-scanning mutants as to putative transmembrane segments 4 and 5 and the flanking regions of Tn10-encoded metal-tetracycline/H ؉ antiporter (TetA (B)) were constructed. All mutants were normally expressed. Among the 57 mutants (L99C to I155C), nine conserved arginine-, aspartate-, and glycine-replaced ones exhibited greatly reduced tetracycline resistance and almost no transport activity, and five conserved glycine-and proline-replaced mutants exhibited greatly reduced tetracycline transport activity in inverted membrane vesicles despite their high or moderate drug resistance. All other cysteine-scanning mutants retained normal drug resistance and normal tetracycline transport activity except for the L142C and I143C mutants. The transmembrane (TM) regions TM4 and TM5 were determined to comprise 20 amino acid residues, Leu-99 to Ile-118, and 17 amino acid residues, Ala-136 to Ala-152, respectively, on the basis of N-[ 14 C]ethylmaleimide ([ 14 C]NEM) reactivity. The NEM reactivity patterns of the TM4 and TM5 mutants were quite different from each other. TM4 could be divided into two halves, that is, a NEM nonreactive periplasmic half and a periodically reactive cytoplasmic half, indicating that TM4 is tilted toward a water-filled transmembrane channel and that only its cytoplasmic half faces the channel. On the other hand, NEM-reactive mutations were observed periodically (every two residues) along the whole length of TM5. A permeability barrier for a membrane-impermeable sulfhydryl reagent, 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid, was present in the middle of TM5 between Leu-142 and Gly-145, whereas all the NEM-reactive mutants as to TM4 were not accessible to 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid, indicating that the channel-facing side of TM4 is located inside the permeability barrier. Tetracycline protected the G141C mutant from the NEM binding, whereas the other mutants in TM4 and TM5 were not protected by tetracycline.

Importance of the GP Dipeptide of the Antiporter Motif and Other Membrane-Embedded Proline and Glycine Residues in Tetracycline Efflux Protein Tet(L)

“…Tet(L) G155C or S retained a modest capacity to confer resistance in the absence of Tc-Co 2+ efflux activity, just as Iwaki et al (23) found for the similarly located G145C mutation of TetA(B) G145C. It is not known whether these findings represent an effect of these mutant Tet proteins on the E. coli host (e.g.…”

“…As with Tet(L) G155C, de-energized control vesicles in transport assays of the TetA(B) G145C mutation (the equivalent position to Tet(L) G155 in the 12-TMS Tc-Me 2+ /H + antiporter) as well as mutants in two other Motif C Gly residues of TetA(B), showed elevated binding compared to controls for other mutants (23). Tet(L) G155C or S retained a modest capacity to confer resistance in the absence of Tc-Co 2+ efflux activity, just as Iwaki et al (23) found for the similarly located G145C mutation of TetA(B) G145C.…”

Identification of molecular hinge points mediating alternating access in the vesicular monoamine transporter VMAT2

“…The spacing between the Gly residues is equivalent to one turn of a helix, indicating a possible structural role. Indeed, Ala or Cys mutants of the second Gly in this motif in TM5 decrease transport activity in some members of the DHA12 family (22,23). Interestingly, in our alignment, motifs in TMs 5 and 11 of rVMAT2 align to Gly-rich segments in the LacY sequence, even though LacY belongs to a different family from the DHA12 proteins.…”

Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA

“…It is likely that Gly-313 may assume a structurally important role that is required for the function of QacA rather than being directly involved in substrate binding. This is commonly seen (10,25,26), as Gly occurs frequently in the TMS of membrane proteins and is often found in interhelical positions (27) that play important roles in mediating interactions between helices (28,29). The lack of a side chain allows Gly to form close interhelical contacts and provides a good packing surface (30); Gly can also be either the donor or acceptor of an interhelical C-H⅐⅐⅐O hydrogen bond that may contribute to the stability of interhelical interactions (31).…”