Studies on the Molecular Structure and Function of Tetracycline Efflux Protein

四环素外流蛋白的分子结构和功能研究

基本信息

批准号：
05454619
负责人：
YAMAGUCHI Akihito
金额：
$ 4.03万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

Since 1990, there have been a lot of drug efflux proteins found in bacteria. So, the molecular biological studies on tetracycline efflux protein (Tet) , which is now a unique protein of which molecular mechanisms are studied, becomes more important as a paradigm of such drug efflux proteins. In this study, we revealed the molecular mechanism of Tet mainly by gene engineering methods.There is a well-known sequence motif widely conserved in secondary membrane transporters such as mammalian glucose transporters, bacterial sugar/H^+ symporters and bacterial drug exporters, which is depicted as GXXSDRXGRR.However, the precise role of this motif in the transporter function is still unknown. We first studied the role of this motif in this study by using site-directed mutagenesis technique. As a result, the 5th Asp66 and 9th Arg70 are essential for the function. As to Arg70, Lys70 mutant retained about 30% the wild type transport activity, whereas the mutants to a neutral or acidic residues lost the transport activity except for Cys70 mutant. Cys70 mutant retained the activity comparable to Lys70 mutant. The unexpectedly high activity of Cys70 mutant is due to the mercaptide formation with a divalent cation which acts as a cationic side chain.Among 8 Cys mutants of this motif, only Cys65 and Cys70 mutants were inactivated by NEM.The binding of NEM to these mutants was stimulated by addition of tetracycline, indicating that the motif, which is located on the cytoplasmic surface, is exposed to the medium by the substrate. In contrast, NEM binding to Cys97, which is located on the periplasmic surface, was inhibited by tetracycline, indicating that the periplasmic residue is hidden by a substrate-induced conformational change. These observations confirm our model for tetracycline/H^+ antiport, in which tetracycline makes Tet protein an inside-open/outside-closed conformation.

自1990年以来，细菌中发现了很多药物外排蛋白。因此，对四环素外排蛋白（TET）的分子生物学研究（现在是一种独特的蛋白质，研究了分子机制）变得越来越重要，因为这种药物外排蛋白的范式变得更加重要。在这项研究中，我们揭示了主要通过基因工程方法的TET的分子机制。这是一个众所周知的序列基序，在次生膜转运蛋白（例如哺乳动物葡萄糖转运蛋白），细菌糖/H^+ sommorporters和细菌药物导出蛋白中广泛保守的序列基序，它被描述为gxxsdrxgrr.snyters nimpers the Motifing shotters notift in the inst shorif. inst sytif ins the inst synter。我们首先使用定点诱变技术研究了这项研究在这项研究中的作用。结果，第五ASP66和9th ARG70对于该函数至关重要。至于ARG70，Lys70突变体保留了约30％的野生型运输活性，而突变体为中性或酸性残基丢失了运输活性，除了Cys70突变体。 Cys70突变体保留了与Lys70突变体相当的活性。 The unexpectedly high activity of Cys70 mutant is due to the mercaptide formation with a divalent cation which acts as a cationic side chain.Among 8 Cys mutants of this motif, only Cys65 and Cys70 mutants were inactivated by NEM.The binding of NEM to these mutants was stimulated by addition of tetracycline, indicating that the motif, which is located on the cytoplasmic surface, is exposed to基板的介质。相比之下，四环素抑制了位于周质表面上的NEM与Cys97的结合，表明周质残基被底物诱导的构象变化隐藏。这些观察结果证实了我们的四环素/H^+对替代模型，其中四环素使TET蛋白成为内部开放/外闭合构象。