Mechanism of metal-specific ECF transporters

能量偶联因子(ECF)转运蛋白是三磷酸腺苷结合盒(ABC)转运蛋白的一个大亚类，参与原核生物对微量营养素的摄取。底物由水溶性维生素和过渡金属离子Co2+和Ni2+组成。所有ECF转运体的共同组成包括一个底物结合的整膜蛋白(S)和由另一个完整的膜蛋白(T)和两个ABC ATPase组成的ECF，最初是在2006年为金属特异的系统描述的。在接下来的几年里，维生素特异性的ECF转运体成为实验研究的重点。最近的结果包括发现序列相似性可以忽略不计的不同底物的S单元具有非常相似的三维拓扑结构，以及底物跨脂双层转运需要S单元在膜内倾倒的结论/假设。金属特异性S单位的第一个3D结构显示了与维生素特异性单位非常相似的拓扑结构。先前的发现证实了额外的N-末端跨膜螺旋在金属特异性S单位中的特定作用。N-末端深入到底物结合口袋，阻止较大分子的进入，并提供方形平面配位场的四种金属配体中的三种。关于(I)N-末端在金属结合过程中如何正确定位，(Ii)如何从结合口袋中取出以将金属释放到细胞质中，以及(Iii)辅助蛋白如何控制这些过程的问题是完全开放的，辅助蛋白具有基本的但未被表征的作用，并且仅存在于金属特异的ECF转运体中。这些问题都在提案的重点范围内。在大量前期工作的基础上，我们旨在利用光谱和生化技术来阐明金属结合和金属释放的机制，以及S单位和辅助蛋白潜在的构象变化。本研究对了解ECF转运蛋白的作用机制具有普遍意义。金属和维生素转运蛋白的S组分、T组分和ATPase组分的相似性表明，它们的转运机制基本相似，但底物装载和释放步骤存在显著差异。

Energy-coupling factor (ECF) transporters represent a large subgroup of the ATP-binding cassette (ABC) transporters and are involved in micronutrient uptake in prokaryotes. Substrates comprise water-soluble vitamins and the transition metal ions Co2+ and Ni2+. The common composition of all ECF transporters consisting of a substrate-binding integral membrane protein (S) and the ECF, composed of another integral membrane-protein (T) and two ABC ATPases, was originally described for metal-specific systems in 2006. In the following years, vitamin-specific ECF transporters were in the focus of experimental research. Recent results include the finding that S units for different substrates with negligible sequence similarity have very similar 3D topologies, and the conclusion/hypothesis that toppling over of S units within the membrane is required for substrate translocation across the lipid bilayer. The first 3D structure of a metal-specific S unit revealed a very similar topology compared to the vitamin-specific counterparts. Previous findings indicating a specific role for the additional N-terminal transmembrane helix in metal-specific S units were confirmed. The very N-terminal end extends deeply into the substrate-binding pocket, blocks access of larger molecules and provides three of the four metal ligands of the square planar ligand field. Questions of (i) how the N-terminus is positioned correctly during metal binding, (ii) how it is removed from the binding pocket for metal release into the cytoplasm, and (iii) how these processes are controlled by a helper protein, that has an essential but uncharacterized role and occurs only in metal-specific ECF transporters, are completely open. Those questions are within the focus of the proposal. Based on extensive preliminary work, we aim at elucidating the mechanism of metal binding and metal release and the underlying conformational changes of the S unit and the helper protein using spectroscopical and biochemical techniques. This research is of general interest for an understanding of the mechanism of ECF transporters. The distinct similarity among S, T and ATPase components of metal- and vitamin-specific ECF transporters suggests a basically similar transport mechanism but significant differences in the substrate-loading and -release steps.