Molecular interaction and high-resolution X-ray structures of the subunits of the osmoregulated ABC-transporter OpuA from B. subtillis

渗透保护剂摄取系统Opua是枯草芽孢杆菌的三个渗透保护性转运蛋白之一，它介导亲和溶质甘氨酸甜菜碱的摄取，甜菜碱是细菌、植物甚至人类细胞广泛使用的渗透保护剂。转运蛋白复合体由一个脂质锚定底物结合蛋白(OpuAC)、一个完整的跨膜蛋白(OpuAB)和一个ATPase亚单位(OpuAA)组成，属于ABC转运蛋白超家族。近年来，渗透保护性转运蛋白复合体Opua在遗传和生理水平上得到了广泛的研究。然而，还没有进行过包括结构、生化或生物物理研究在内的体外研究。基于我们到目前为止所进行的生化研究，我们建议从其结构和复合体内各亚单位之间的通讯的动态方面来研究这个转运蛋白复合体。Opua系统的体外和体内实验将伴随着我们的结构研究。对于体外实验，结晶学、表面等离子体共振和荧光技术将是重要的。这些研究将得到体内实验的帮助，这些实验使用已经存在的枯草杆菌渗透调节转运蛋白系统的缺失菌株。基于所提出的实验，我们希望对枯草杆菌Opua ABC转运蛋白的结构要求和通讯机制提供详细的了解。

The osmoprotectant uptake system OpuA is one of the three osmoprotective transporters in Bacillus subtilis, which mediate the uptake of the compatible solute glycine betaine, an osmoprotectant widely used by bacteria, plants and even human cells. The transporter complex consists of a lipid-anchored substrate-binding protein (OpuAC), an integral transmembrane protein (OpuAB), and an ATPase subunit (OpuAA) and belongs to the super family of ABC-transporters. In recent years, the osmoprotective transporter complex OpuA has been intensively characterized on a genetic and physiological level. However, no in vitro studies have been performed including structural, biochemical or biophysical studies. Based on our biochemical studies performed so far, we propose to study this transporter complex with respect to its structure and the dynamic aspects of the communication between the subunits within the complex. In vitro and in vivo experiments with the OpuA system will accompany our structural investigations. For the in vitro experiments, crystallography, surface plasmon resonance, and fluorescence techniques will be of major importance. These studies will be aided by in vivo experiments using already available deletion strains of the osmo-regulated transporter systems of B. subtilis. Based on the proposed experiments, we hope to provide a detailed understanding of the structural requirements and communication mechanisms governing the function of the OpuA ABC-transporter of B. subtilis.