Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: Identification of a conserved motif
Biochemical characterization of domains in the membrane subunit DrrB that interact with the ABC subunit DrrA: Identification of a conserved motif
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DOI：
10.1021/bi048959c
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发表时间：
2005-02-22
期刊：
影响因子：
2.9
通讯作者：
Gandlur, SM
中科院分区：
文献类型：
--
作者：
Kaur, P;Rao, DK;Gandlur, SM
DrrA和DrrB蛋白质赋予生产者生物链霉菌Peucetius对常用抗癌剂柔红霉素和多柔比星的抗性。drrAB基因座先前已在大肠杆菌中克隆，并且已发现蛋白质在该宿主中具有功能。DrrA是一种可溶性蛋白，属于ABC蛋白家族。它与整合的膜蛋白DrrB形成复合物。以往的研究表明，DrrA和DrrB的功能和稳定性是生物化学耦合的。因此，DrrA仅在与膜中的DrrB形成复合物时才与ATP结合。此外，如果DrrA不存在，则DrrB完全降解。在本研究中，我们已经表征了可能直接参与与DrrA相互作用的DrrB结构域。在DrrB中进行了几个单半胱氨酸取代。DrrA和DrrB之间的相互作用进行了研究，通过使用半胱氨酸胺化学交联剂，特异性交联一个蛋白质（DrrB）中的游离巯基在另一个（DrrA）的胺。我们在这里表明，DrrA交联与N-和C-末端的DrrB蛋白，这意味着它们可能参与相互作用。此外，这项研究确定了一个基序的N-末端胞质尾的DrrB，这是类似的基序最近显示的晶体结构分析在BtuC和以前显示的序列分析也存在于出口商，包括MDR 1。我们建议，目前在DrrB和其他出口商的基序实际上是一个修改后的版本的EAA基序，这最初被认为是目前只在进口商的ABC家庭。目前的工作是第一份报告，在ABC药物出口商的膜组件中的相互作用域已被生化表征。
DrrA and DrrB proteins confer resistance to the commonly used anticancer agents daunorubicin and doxorubicin in the producer organism Streptomyces peucetius. The drrAB locus has previously been cloned in Escherichia coli, and the proteins have been found to be functional in this host. DrrA, a soluble protein, belongs to the ABC family of proteins. It forms a complex with the integral membrane protein DrrB. Previous studies suggest that the function and stability of DrrA and DrrB are biochemically coupled. Thus, DrrA binds ATP only when it is in a complex with DrrB in the membrane. Further, DrrB is completely degraded if DrrA is absent. In the present study, we have characterized domains in DrrB that may be directly involved in interaction with DrrA. Several single-cysteine substitutions in DrrB were made. Interaction between DrrA and DrrB was studied by using a cysteine to amine chemical cross-linker that specifically cross-links a free sulfhydryl group in one protein (DrrB) to an amine in another (DrrA). We show here that DrrA cross-links with both the N- and the C-terminal ends of the DrrB protein, implying that they may be involved in interaction. Furthermore, this study identifies a motif within the N-terminal cytoplasmic tail of DrrB, which is similar to a motif recently shown by crystal structure analysis in BtuC and previously shown by sequence analysis to be also present in exporters, including MDR1. We propose that the motif present in DrrB and other exporters is actually a modified version of the EAA motif, which was originally believed to be present only in the importers of the ABC family. The present work is the first report where domains of interaction in the membrane component of an ABC drug exporter have been biochemically characterized.