Mf. Varela et al., MUTATIONAL ANALYSIS AND MOLECULAR MODELING OF AN AMINO-ACID-SEQUENCE MOTIF CONSERVED IN ANTIPORTERS BUT NOT SYMPORTERS IN A TRANSPORTER SUPERFAMILY, Molecular membrane biology, 12(4), 1995, pp. 313-319
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.