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Studies on the Bacterial Xenobiotic Efflux Mechanism

细菌异生物质外排机制的研究

基本信息

批准号：
08457604
负责人：
YAMAGUCHI Akihito
金额：
$ 4.93万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

Bacterial metal-tetracycline/H^+ antiporter (Tet (B)) is one of typical xenobiotic exporters which have been widely found in living organisms as basic devices for host defense mechanism. In this study, we investigated the molecular structure of Tet (B) and the pathway of the drug extrusion. Cysteine-scanning mutants of Tet (B) were constructed on the basis of the Cys-free mutant. When the Cys residue is located on the outside of the membrane, the binding of membrane-permeable SH reagent, [14C]NEM,to the Cys residue of Tet (B) expressed in the intact cells should be competitively inhibited by an membrane-impermeable reagent, AMS.On the contrary, when it is located inside, the binding should not be inhibited. By means of this method, we experimentaly proved the 12 membrane-spanning structure of Tet (B). Then, we succeeded to determine the exact boundary between the membrane-spanning segments and the water-extruding loops by means of the reactivity of [14C] NEM with the Cys-scanning mutants. The Cys residues located in the membrane-spnanning reagion showed no or very low reactivity with NEM,however, the Cys residues on the water-exposed loops showed high reactivity. The confomational change during the substrate translocation or by the mutation was also detected by means of the reactivity of [14C] NEM with Cys residues : (1) Tet (B) underwent the substrate-induced conformational change from the inside-closed/outside-open conformation to the inside-open/outside-closed one, and (2) the remote-conformational change caused by a mutation was suppressed by a second-site suppressor mutation. On the basis of these protein-engineering studies, we proposed the 3D model of Tet (B) protein.

细菌金属-四环素/H~+逆向转运蛋白(Tet(B))是一种典型的异源输出体，广泛存在于生物体中，是机体防御机制的基本装置。在本研究中，我们研究了Tet(B)的分子结构和药物挤出的途径。Tet(B)的半胱氨酸扫描突变体是在无半胱氨酸突变体的基础上构建的。当Cys残基位于膜外时，膜透性SH试剂[14C]NEM与在完整细胞中表达的Tet(B)的Cys残基的结合应被膜不通透性试剂AMS竞争性抑制。相反，当它位于膜内时，结合不受抑制。用这种方法，我们实验证明了Tet(B)的12个跨膜结构。然后，我们通过[14C]NEM与半胱氨酸扫描突变体的反应，成功地确定了膜跨段和水挤出环之间的准确边界。膜反应中的半胱氨酸残基与NEM无反应性或很低反应性，而水暴露环上的半胱氨酸残基表现出很高的反应性。还通过[14C]NEM与半胱氨酸残基的反应检测了底物易位或突变引起的构象变化：(1)Tet(B)经历了底物诱导的从内封闭/外开放构象到内开放/外封闭构象的变化，(2)突变引起的远端构象变化被第二位点抑制突变所抑制。在这些蛋白质工程研究的基础上，我们提出了Tet(B)蛋白质的三维模型。