Studies on the Bacterial Xenobiotic Efflux Mechanism

细菌金属四环素/H^+逆向转运蛋白（泰特（B））是一种典型的外源性物质输出蛋白，广泛存在于生物体内，是宿主防御机制的基本装置。本研究对泰特（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泰特（B）的半胱氨酸扫描突变体。当Cys残基位于膜外时，膜渗透性SH试剂[14 C]NEM与完整细胞中表达的泰特（B）的Cys残基的结合应受到膜渗透性试剂AMS的竞争性抑制。相反，当其位于膜内时，结合不应受到抑制。用这种方法，我们从实验上证明了泰特（B）的十二跨膜结构。然后，我们成功地确定了跨膜段和水挤出环之间的确切边界，通过[14 C] NEM与Cys扫描突变体的反应性。位于跨膜区的Cys残基与NEM无反应或反应性很低，而暴露于水的环上的Cys残基与NEM反应性很高。还通过[14 C] NEM与Cys残基的反应性检测了底物移位过程中或突变引起的构象变化：（1）泰特（B）经历了底物诱导的构象变化，从内封闭/外开放构象转变为内开放/外封闭构象，（2）突变引起的远程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，我们提出了泰特（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.