Dynamics of subunit interactions in ECF transporters

能量偶联因子“转运体由底物专一性(S)和适度保守(T)的完整膜蛋白和ABC-ATPase组成。与经典的ABC进口体不同，ECF系统不依赖于细胞质外溶质结合蛋白。A-、T-和S-单位的低聚组成，S和T-单位的确切作用，以及三磷酸腺苷水解过程中的动态相互作用与底物易位的分子机制分别存在争议或未知。晶体结构分析确定了三种不同的S单元为底物负载单体。另一方面，活体研究、定点突变和交联实验支持S的低聚状态，并可能将T单位作为体内的功能状态。本申请的重点是生物素转运蛋白(BioYMN)。我们以前的结果倾向于将生物二聚体作为具有功能的S单位，它在其孤立状态下具有转运(除了底物结合)活性。一个合适的大肠杆菌报告菌株将被用来最终通过一组Bioy同源物来澄清有争议的Bioy运输功能的问题。T单位Bion和Bioy的低聚状态将通过使用活细胞和重组在纳米盘中的纯化转运蛋白复合体的荧光光谱和成像技术进行分析。EPR技术将作为补充方法应用于纳米盘中的自旋标记络合物。通过动力学分析、下拉实验和EPR实验研究了ATP水解过程中亚基相互作用的动力学。一种正在进行的外部合作的结构生物学方法旨在解决ECF全转运体的3D结构，作为了解亚单位组织的重要一步。

Energy-coupling factor (ECF)" transporters are composed of a substrate-specific (S) and a moderately conserved (T) integral membrane protein and of ABC ATPases. In contrast to canonocal ABC importers, ECF systems do not rely on extracytoplasmic solute-binding proteins. The oligomeric composition of A-, T- and S-units, the exact roles of the S- and T-components and the molecular mechanisms that couple dynamic interactions during ATP hydrolysis to substrate translocation are controversial or unknown, respectively. Crystal structure analysis identified three different S-units as substrate-loaded monomers. In vivo investigations, site-directed mutagenesis and crosslinking experiments, on the other hand, favor oligomeric states of the S- and potentially the T-units as the functional in vivo states. The focus of the present application is on biotin transporters (BioYMN). Our previous results favor a BioY dimer as the functional S-unit that has transport (in addition to substrate-binding) activity in its solitary state. An appropriate E. coli reporter strain will be used to clarify eventually the controversially discussed issue of BioY's transport function using a set of BioY homologs. The oligomeric states of the T-unit BioN and of BioY will be analyzed by fluorescence-spectroscopical and –imaging techniques using live cells as well as purified transporter complexes reconstituted in nanodiscs. EPR techniques will be applied to spin-labeled complexes in nanodiscs as a complementary approach. The dynamics of subunit interactions during ATP hydrolysis will be investigated by kinetic analysis, pull-down and EPR experiments. An ongoing structural biology approach in external collaboration aims at solving the 3D structure of an ECF holotransporter as a major step towards an understanding the subunit organization.