Mutational analysis and molecular modelling of an amino acid sequence motif conserved in antiporters but not symporters in a transporter superfamily.
Elements of a 'G X8 G X3 G P X2 G G' amino acid sequence motif were conserved in the fifth predicted membrane-spanning domains of 31 antiporters, but none of 27 symporters or uniporters that together comprise a 'superfamily' of structurally, related transport proteins. Molecular modelling and mechanics predicted that the GP dipeptide of this motif bends the antiporters' fifth transmembrane helices, and that the repeating pattern of glycine residues forms a pocket, devoid of side chains, on the surface of these helices. The glycine residue in the motif's GP dipeptide was conserved in 90% of these antiporters with alanine being the only observed substitution. Replacement of the glycine residue of the GP dipeptide with alanine and serine reduced the level of tetracycline resistance conferred by TetA(C), a tetracycline/H+ antiporter, 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 susceptibility 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 activity of TetA(C), rather than its expression, stability, or general three-dimensional structure.
Varela
M F
MF
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Sansom
C E
CE
Griffith
J K
JK
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D017920:Antiporters / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002104:Cadmium / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics; D002462:Cell Membrane / Q000737:chemistry; D016384:Consensus Sequence; D004252:DNA Mutational Analysis; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011387:Prokaryotic Cells / Q000378:metabolism; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013753:Tetracycline Resistance
D000596:Amino Acids; D017920:Antiporters; D001426:Bacterial Proteins; D002352:Carrier Proteins; C079051:tetA protein, Bacteria; D002104:Cadmium