Varela M F, Sansom C E, Griffith J K
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131, USA.
Mol Membr Biol. 1995 Oct-Dec;12(4):313-9. doi: 10.3109/09687689509072433.
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.
在31种反向转运蛋白的第五个预测跨膜结构域中，“G X8 G X3 G P X2 G G”氨基酸序列基序的元件是保守的，但在总共构成一个结构相关转运蛋白“超家族”的27种同向转运蛋白或单向转运蛋白中则没有保守。分子建模和力学预测，该基序的GP二肽会使反向转运蛋白的第五个跨膜螺旋弯曲，并且甘氨酸残基的重复模式在这些螺旋的表面形成了一个没有侧链的口袋。在这些反向转运蛋白中，90%的基序GP二肽中的甘氨酸残基是保守的，唯一观察到的替代物是丙氨酸。用丙氨酸和丝氨酸替代GP二肽中的甘氨酸残基，分别使四环素/H⁺反向转运蛋白TetA(C)赋予的四环素抗性水平降低了74%和81%。所有其他替代物完全消除了对四环素的抗性。相比之下，替代GP二肽中的甘氨酸残基并没有消除对镉的敏感性增加，镉敏感性增加是TetA(C)赋予的另一种表型，与对四环素的抗性无关。这些结果表明，GP二肽中的甘氨酸对于TetA(C)的四环素/H⁺反向转运活性是必需的，而不是其表达、稳定性或总体三维结构所必需的。
