From the Departments of Biology and.
Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892; Chemistry, Catholic University of America, Washington, D. C. 20064.
J Biol Chem. 2013 Oct 18;288(42):30420-30431. doi: 10.1074/jbc.M113.494682. Epub 2013 Sep 9.
Pdr5 is the founding member of a large subfamily of evolutionarily distinct, clinically important fungal ABC transporters containing a characteristic, deviant ATP-binding site with altered Walker A, Walker B, Signature (C-loop), and Q-loop residues. In contrast to these motifs, the D-loops of the two ATP-binding sites have similar sequences, including a completely conserved aspartate residue. Alanine substitution mutants in the deviant Walker A and Signature motifs retain significant, albeit reduced, ATPase activity and drug resistance. The D-loop residue mutants D340A and D1042A showed a striking reduction in plasma membrane transporter levels. The D1042N mutation localized properly had nearly WT ATPase activity but was defective in transport and was profoundly hypersensitive to Pdr5 substrates. Therefore, there was a strong uncoupling of ATPase activity and drug efflux. Taken together, the properties of the mutants suggest an additional, critical intradomain signaling role for deviant ATP-binding sites.
Pdr5 是一个大型进化上不同的、具有临床重要性的真菌 ABC 转运蛋白亚家族的创始成员，该亚家族包含一个特征性的、异常的 ATP 结合位点，其 Walker A、Walker B、Signature（C 环）和 Q 环残基发生了改变。与这些基序相反，两个 ATP 结合位点的 D 环具有相似的序列，包括一个完全保守的天冬氨酸残基。异常 Walker A 和 Signature 基序中的丙氨酸取代突变体保留了显著的（尽管降低了）ATP 酶活性和耐药性。D 环残基突变体 D340A 和 D1042A 显示出明显减少的质膜转运蛋白水平。正确定位的 D1042N 突变体具有几乎 WT 的 ATP 酶活性，但在转运中存在缺陷，并且对 Pdr5 底物表现出严重的超敏反应。因此，ATP 酶活性和药物外排之间存在强烈的解偶联。总之，突变体的特性表明异常的 ATP 结合位点具有额外的、关键的域内信号转导作用。
