MUTATIONAL ANALYSIS AND MOLECULAR MODELING OF AN AMINO-ACID-SEQUENCE MOTIF CONSERVED IN ANTIPORTERS BUT NOT SYMPORTERS IN A TRANSPORTER SUPERFAMILY

Elements of a 'G X(8) G X(3) G P X(2) G G' amino acid sequence motif w ere conserved in the fifth predicted membrane-spanning domains of 31 a ntiporters, but none of 27 symporters or uniporters that together comp rise a 'superfamily' of structurally-related transport proteins. Molec ular modelling and mechanics predicted that the GP dipeptide of this m otif bends the antiporters' fifth transmembrane helices, and that the repeating pattern of glycine residues forms a pocket, devoid of side c hains, on the surface of these helices. The glycine residue in the mot if's GP dipeptide was conserved in 90% of these antiporters with alani ne being the only observed substitution. Replacement of the glycine re sidue of the GP dipeptide with alanine and serine reduced the level of tetracycline resistance conferred by TetA(C), I a tetracycline/H+ ant iporter, by 74 and 81%, respectively. All other substitutions totally abolished resistance to tetracycline. In contrast, replacement of the glycine residue of the GP dipeptide did not abolish increased suscepti bility to cadmium, another phenotype conferred by TetA(C) independent of resistance to tetracycline. These results suggest that the glycine of the GP dipeptide is necessary for the tetracycline/H+ antiport acti vity of TetA(C), rather than its expression, stability, or general thr ee-dimensional structure.