SITE-SPECIFICITY OF THE SECOND-SITE SUPPRESSOR MUTATION OF THE ASP-285-]ASN MUTANT OF METAL-TETRACYCLINE/H+ ANTIPORTER OF ESCHERICHIA-COLI AND THE EFFECTS OF AMINO-ACID SUBSTITUTIONS AT THE FIRST AND 2ND SITES

The deleterious effect of the mutation of Asp-285 to Asn of the metal-tetracycline/H+ antiporter (TetA) of Escherichia coli is suppressed by the second-site mutation of Ala-220 to an acidic amino acid residue (Yamaguchi, A., O'yauchi, R., Someya, Y., & Sawai, T. (1993) J. Biol. Chem. 268, 26990-26995). In this study, site-specific second-site Glu mutants as to 11 different positions around position 220 were established from the Asp-285 --> Asn mutant TetA protein. Among them, only the Ala-220 Glu mutant completely suppressed the deleterious effect of the Asp-285 --> Asn mutation, indicating that position 220 is highly specific for the suppression. Although E. coli cells producing second-site Glu mutants as to positions 213, 216, 217, 218, 219, 221, 222, and 223 of the Asn-285 mutant were as tetracycline sensitive as the host cells without TetA, Gly-224 --> Glu and Pro-227 --> Glu second-site mutants of the Asn-285 mutant conferred low-level tetracycline resistance, the levels decreasing in this order. These two positions and position 220 are on the same side of putative transmembrane helix VII. The Phe-289 --> Asp mutation, which is located at a position one-alpha-helical-turn downstream from Asp-285 in the same putative helix, IX, did not suppress the Asn-285 mutation. The introduction of an acidic residue at the second site was essential for suppression of the Asn-285 mutation because Lys-220 and Gln-220 second-site mutants of the Asn-285 mutant showed very low tetracycline resistance. Interestingly, the second-site Glu or Asp mutation at position 220 showed no suppression of the Asp-285 --> Lys mutation, indicating that the positive charge at position 285 inhibits the compensatory function of the carboxylic acid side chain at position 220, probably due to an ionic interaction. These results revealed that the essential function of Asp-285 is specifically compensated for by a newly introduced carboxylic acid side chain around position 220 on one side of putative helix VII, which is sterically close to position 285 on putative helix IX.