Role of the Charge Interaction between Arg70and Asp120in the Tn10-encoded Metal-Tetracycline/H+Antiporter ofEscherichia coli

Yuichi Someya, Tomomi Kimura-Someya, Akihito Yamaguchi
2000 Journal of Biological Chemistry  
We reported that the positive charge of Arg 70 is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H ؉ antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Arg 70 may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp 66 and Asp 120 , which are only two negatively charged residues located close to Arg 70 in putative secondary structure of TetA(B) and highly conserved
more » ... nd highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp 66 is essential, but Asp 120 is important for TetA(B) function. Surprisingly, when Asp 120 was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp 66 mutation. The chargeexchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg 2؉ ), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg 2؉ ; however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg 2؉ , probably because the side chains at positions 70 and 120 are bridged with Hg 2؉ . The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10phenanthroline). These results indicate that the positive charge of Arg 70 requires the negative charge of Asp 120 for neutralization, probably for properly positioning transmembrane segments in the membrane. TetA(B), class B TetA protein; NEM, N-ethylmaleimide; MOPS, 4-morpholinepropanesulfonic acid.
doi:10.1074/jbc.275.1.210 pmid:10617606 fatcat:646oo5oomnfp5iz5kdd6majuvq