From the Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany.
From the Institute of Biochemistry, Heinrich Heine University Düsseldorf, Universitätsstrasse 1, 40225 Düsseldorf, Germany
J Biol Chem. 2014 May 30;289(22):15272-9. doi: 10.1074/jbc.M114.553065. Epub 2014 Apr 14.
Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely "regenerated" Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.
Pdr5 是一种来自酿酒酵母的质膜结合 ABC 转运蛋白，参与了对临床相关的细菌、真菌和人类的外源物质的抗性现象。许多真菌 ABC 转运蛋白，如 Pdr5，在其核苷酸结合位点（NBS）中表现出固有不对称性，这与大多数人类对应物不同。这种 NBS 的简并性非常有趣，需要从结构和功能相关性的角度来解释。在这项研究中，我们将 NBS 中的非共识氨基酸残基突变为其共识对应物，并研究了其对蛋白质功能和对酵母细胞的影响。完全“再生”的 Pdr5 蛋白在其 ATP 水解和罗丹明 6G 转运功能上严重受损。此外，我们还观察到一些突变体中存在替代的补偿机制，以对抗药物毒性。从本质上讲，我们在这里首次尝试在不对称 ABC 转运蛋白中恢复完全对称性，并研究其可能与整个不对称 ABC 转运蛋白类相关的影响。
