Mechanism of metal-specific ECF transporters

能量偶联因子（ECF）转运体是atp结合盒（ABC）转运体的一个大亚群，并参与原核生物微量营养素的摄取。底物包括水溶性维生素和过渡金属离子Co2+和Ni2+。所有ECF转运体的共同组成包括底物结合的整体膜蛋白(S)和由另一个整体膜蛋白(T)和两个ABC atp酶组成的ECF，最初在2006年被描述为金属特异性系统。在接下来的几年里，维生素特异性ECF转运体成为实验研究的重点。最近的研究结果包括发现不同底物的S单元具有可忽略不计的序列相似性，具有非常相似的3D拓扑结构，并且结论/假设膜内S单元的翻转是底物跨脂质双分子层易位所必需的。金属特异性S单元的第一个3D结构显示出与维生素特异性S单元非常相似的拓扑结构。先前的研究结果表明，额外的n端跨膜螺旋在金属特异性S单元中的特定作用得到了证实。极n端深入底物结合袋，阻断大分子的进入，并提供方形平面配体场中四个金属配体中的三个。(i)在金属结合过程中n端是如何正确定位的，（ii）它是如何从结合口袋中移除金属释放到细胞质中的，以及（iii）这些过程是如何由辅助蛋白控制的，该蛋白具有重要但未表征的作用，仅发生在金属特异性ECF转运蛋白中，这些问题都是完全开放的。这些问题是提案的重点。基于广泛的前期工作，我们旨在利用光谱和生化技术阐明金属结合和金属释放的机制以及S单元和辅助蛋白的潜在构象变化。这项研究对了解ECF转运体的机制具有普遍的意义。金属特异性ECF转运体和维生素特异性ECF转运体的S、T和atp酶组分之间存在明显的相似性，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.