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

Abstract

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)). In 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.

Cysteine‐scanning mutagenesis around transmembrane segment VI of Tn10‐encoded metal‐tetracycline/H+ antiporter

“…All mutants showed the same resistance as the wild‐type TetA(B) (200 μg/ml) except for the S156C mutant, which showed moderate resistance (25 μg/ml), indicating that there are no functionally essential residues in this region. This observation is consistent with the previous conclusion that the suppression of the mutation of Arg‐70 by the second‐site mutation of Thr‐171 was not due to a functional substitution but due to a remote‐conformational distortion [11].…”

“…In this study, we chose transmembrane helix VI for cysteine‐scanning mutagenesis. The presence of a close conformational linkage between this transmembrane helix and the putative cytoplasmic gating region, loop 2‐3, was predicted because the second‐site mutation at Thr‐171, which is located in the middle of TM VI, suppresses the first charge‐neutralized mutation of Arg‐70, which is a functionally important residue located in the putative gating region, loop 2‐3 [11].…”

Role of the Charge Interaction between Arg70 and Asp120 in the Tn10-encoded Metal-Tetracycline/H+ Antiporter of Escherichia coli

“…The difference between lactose permease and TetA(B) (and also ␣-ketoglutarate permease) may be due to the difference in the amino acid sequence of the motif. However, TetA(B) and lactose permease share the property that the loop 2-3 region of two transporters is important for conformational change of the protein during the transport cycle (33,(35)(36)(37)(38)(39).…”

Site-directed Chemical Modification of Cysteine-scanning Mutants as to Transmembrane Segment II and Its Flanking Regions of the Tn10-encoded Metal-Tetracycline/H+ Antiporter Reveals a Transmembrane Water-filled Channel

“…The first and eighth Gly, fifth Asp, and ninth Arg were revealed to be important for the tetracycline transport function or maintenance of the protein conformation (24). We isolated second-site suppressor mutants of the G62L, D66C, and R70A mutants (25)(26)(27). The second-site mutations of the former two mutants (G62L and D66C) occurred in the first periplasmic loop region, loop1-2 (25,26).…”