Remote Conformational Effects of the Gly-62 → Leu Mutation of the Tn10-Encoded Metal-Tetracycline/H+ Antiporter of Escherichia coli and Its Second-Site Suppressor Mutation

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Abstract
Substitution of Gly-62 of the Tn10-encoded metal-tetracycline/H+ antiporter caused a functional defect corresponding to the volume of the substituent [Yamaguchi, A., Someya, Y., and Sawai, T. (1992) J. Biol. Chem. 267, 19155-19162]. A spontaneous revertant exhibiting tetracycline resistance was isolated from Escherichia coli cells carrying the tetA(B) gene encoding the G62L mutation. The revertant showed a second-site mutation at codon 30 of TTA (Leu) to TCA (Ser). Site-directed mutagenesis studies revealed that the L30S mutation could suppress the effects of the G62A, G62C, G62N, and G62V mutations as well as the G62L mutation. Positions 62 and 30 are located in hydrophilic loops estimated to be in the cytoplasm and periplasm, respectively. Their sidedness was confirmed by the fact that, in intact cells, the [14C]N-ethylmaleimide (NEM) binding to the G62C mutant was not affected by preincubation with a membrane-impermeant sulfhydryl reagent, whereas the binding to the L30C mutant was blocked by the reagent. The reactivity of the L30C and L29C mutants with [14C]NEM was drastically decreased when Gly-62 was replaced by Leu, indicating that the residues around position 30 became embedded in the intramembrane region due to the remote conformational effect of the G62L mutation across the membrane. Moreover, the reactivity of the L29C/G62L mutant with [14C]NEM was restored with the L30S mutation. These results clearly indicate that the second-site suppression by the L30S mutation was based on the blocking of the remote conformational change around position 30 across the cell membrane caused by the G62L mutation.