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，在细菌中发现了大量的药物外排蛋白。因此，四环素外排蛋白（Tet）的分子生物学研究作为此类药物外排蛋白的范例变得更加重要，四环素外排蛋白（Tet）现在是一种研究其分子机制的独特蛋白质。本研究主要通过基因工程方法揭示了Tet的分子机制。在哺乳动物葡萄糖转运蛋白、细菌糖/H^+同向转运蛋白和细菌药物输出蛋白等次级膜转运蛋白中，存在着广泛保守的众所周知的序列基序，该序列基序广泛存在于次级膜转运蛋白中，该序列基序广泛保守于次级膜转运蛋白中。被描述为 GXXSDRXGRR。然而，该基序在转运蛋白功能中的确切作用仍然未知。我们首先通过使用定点诱变技术研究了该基序在本研究中的作用。因此，第 5 个 Asp66 和第 9 个 Arg70 对于该功能至关重要。对于Arg70，Lys70突变体保留了野生型约30％的转运活性，而中性或酸性残基的突变体则失去了除Cys70突变体之外的转运活性。 Cys70突变体保留了与Lys70突变体相当的活性。 Cys70突变体出人意料的高活性是由于与作为阳离子侧链的二价阳离子形成硫醇。在该基序的8个Cys突变体中，只有Cys65和Cys70突变体被NEM灭活。NEM与这些突变体的结合通过添加四环素来刺激，表明位于细胞质表面的基序通过底物暴露于培养基。相反，NEM 与位于周质表面的 Cys97 的结合被四环素抑制，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^+反向转运模型，其中四环素使 Tet 蛋白成为内开/外闭构象。