Second-site suppressor mutations for the Arg70 substitution mutants of the Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli

The positive charge of the Arg70 residue in the cytoplasmic loop of the Tn10-encoded metal-tetracycline/H+ antiporter (Tet(B)) of Escherichia coli is essential for the tetracycline transport function (Y. Someya and A. Yamaguchi, Biochemistry 35, 9385-9391 (1996)). Ln this study, we found that the R70A mutation was suppressed by the second-site mutation of Thr171 to Ser. The T171S mutation suppressed any mutations at position 70 regardless of the amino acid residue introduced. The R70A and R70C mutations were also suppressed by the T171A or T171C mutations, but not by the T171Y mutation, indicating that the decrease in the side chain volume at position 171 is essential for the suppression Tetracycline transport activity of the R70C mutant was stimulated by Hg2+ because mercaptide formed between the SH group of Cys70 and Hg2+ worked as a functional positively-charged side chain. The activity of the R70A/R71C/T171S mutant was also stimulated by Hg2+, whereas those of the R70A/R71C, R71C, and R71C/T171S mutants were not, indicating that the T171S mutation causes the switching of the functional positive charge at position 70 to 71. Since Thr171 is in the middle of the transmembrane helix VI, the switching may be based on a remote conformational effect. (C) 1997 Elsevier Science B.V.