Studies on the Bacterial Xenobiotic Efflux Mechanism

细菌金属四环素/H^+反转运体（Tet (B)）是一种典型的外源输出体，广泛存在于生物体中，是宿主防御机制的基本装置。本研究对Tet (B)的分子结构和药物挤出途径进行了研究。在无cys突变体的基础上构建了Tet (B)半胱氨酸扫描突变体。当Cys残基位于膜外时，膜透性SH试剂[14C]NEM与完整细胞中表达的Tet (B)的Cys残基的结合应被膜不透性试剂AMS竞争性地抑制。相反，当它位于内部时，不应抑制其结合。通过这种方法，我们实验证明了Tet (B)的12膜跨越结构。然后，我们成功地通过[14C] NEM与cys扫描突变体的反应性确定了跨膜段和水挤出环之间的确切边界。位于跨膜区的Cys残基与NEM的反应性不高或极低，而位于水暴露环上的Cys残基则表现出较高的反应性。通过[14C] NEM与Cys残基的反应性也检测了底物易位或突变时的构象变化：(1)Tet (B)经历了底物诱导的构象变化，从内闭/外开构象变为内开/外闭构象；(2)突变引起的远程构象变化被第二位点抑制基因突变抑制。在这些蛋白质工程研究的基础上，我们提出了Tet (B)蛋白的三维模型。

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.