A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5
A mutation of the H-loop selectively affects rhodamine transport by the yeast multidrug ABC transporter Pdr5
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DOI：
10.1073/pnas.0800191105
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发表时间：
2008-04-01
影响因子：
11.1
通讯作者：
Schmitt, Lutz
中科院分区：
文献类型：
--
作者：
Ernst, Robert;Kueppers, Petra;Schmitt, Lutz
酵母ABC转运蛋白Pdr 5在对大量结构无关化合物的耐药性中起主要作用。虽然Pdr 5已被广泛研究，但关于其分子机制的许多重要方面仍未解决。例如，在核苷酸结合结构域（NBD）中存在保守氨基酸残基的显著变性，但它们的功能相关性尚不清楚。在这里，我们进行了体内和体外实验，以解决NBD的功能不对称性。ATP酶活性和药物转运研究表明，两个NBD复合位点中只有一个位点的催化作用对蛋白质功能至关重要。此外，对提出的“催化羧酸盐”（E1036）和“催化二联体组氨酸”（H1068）的突变进行了表征。虽然谷氨酸的突变废除ATP酶活性和底物转运，突变H1068对ATP消耗没有影响。然而，H1068 A突变废除罗丹明运输在体内和体外，而离开其他基板的运输不受影响。与哺乳动物P-糖蛋白（P-gp）相比，酵母Pdr 5的ATP酶活性不受底物的刺激，表明Pdr 5是一种非偶联的ABC转运蛋白，其不断水解ATP以确保活性底物转运。两者合计，我们的数据提供了重要的见解Pdr 5的分子机制，并表明，不只是跨膜结构域决定底物的选择。
The yeast ABC transporter Pdr5 plays a major role in drug resistance against a large number of structurally unrelated compounds. Although Pdr5 has been extensively studied, many important aspects regarding its molecular mechanisms remain unresolved. For example, a striking degeneration of conserved amino acid residues exists in the nucleotide binding domains (NBDs), but their functional relevance is unknown. Here, we performed in vivo and in vitro experiments to address the functional asymmetry of NBDs. it became evident by ATPase activity and drug transport studies that catalysis at only one of the two NBD composite sites is crucial for protein function. Furthermore, mutations of the proposed "catalytic carboxylate" (E1036) and the "catalytic dyad histidine" (H1068) were characterized. Although a mutation of the glutamate abolished ATPase activity and substrate transport, mutation of H1068 had no influence on ATP consumption. However, the H1068A mutation abolished rhodamine transport in vivo and in vitro, while leaving the transport of other substrates unaffected. By contrast to mammalian P-glycoprotein (P-gp), the ATPase activity of yeast Pdr5 is not stimulated by the addition of substrates, indicating that Pdr5 is an uncoupled ABC transporter that constantly hydrolyses ATP to ensure active substrate transport. Taken together, our data provide important insights into the molecular mechanism of Pdr5 and suggest that not solely the transmembrane domains dictate substrate selection.