Role of lipids on the conformational cycling in trimeric Na+-coupled symporters

次级转运蛋白的功能受到寡聚化的高度影响。一个主要的例子是Na+偶联的甜菜碱转运蛋白BetP，BCCT家族的成员，其仅作为三聚体被调节。在三聚体状态下，发生特异性脂质接触以及末端结构域的三聚体内相互作用，导致单个原聚体相对于膜法线的重新取向。因此，在BetP中Na+耦合的翻转速率和协同性以K+依赖的方式改变。由于结构上不相关，SLC 1家族的Na+/K+/H+偶联谷氨酸转运蛋白（例如EAAT 1）与BetP共享三聚体组装。SLC 1转运蛋白中的中心“三聚化结构域”深深嵌入膜中，使得三个“转运结构域”在交替进入期间能够进行特征性和独立的电梯运动。在BetP中，C-末端结构域是主要的转运调节剂。同样，在EAAT 1和EAAT 2中，显示各自的C-末端结构域改变谷氨酸摄取循环和氯离子渗透性。在本项目中，我们希望研究在三聚体EAAT 1和BetP中是否存在共同的机制模式，通过这些机制模式调节和/或调节脂质相互作用影响Na+偶联和K+反向转运的协同性。这些进化上不相关的三聚体共转运体之间的任何机械相似性可能表明钠偶联运输调节的非常普遍的原则。因此，我们希望在BetP和EAAT 1之间进行比较结构动力学研究。我们希望研究（1）三聚体作为脂质和调节剂相互作用平台的作用，以及（2）K+对BetP和EAAT 1中协同钠偶联的调节作用。为了实现这一目标，我们希望使用这些三聚体转运蛋白，其中广泛的结构信息已经可用于建立结构生物学中的新工具，主要使用低温扫描透射电子显微镜（cryo-STEM）和FTIR模式下的单分子扫描近场光学显微镜（s-SNOM）。通过cryo-STEM，我们希望分配cryo-EM结构中的K+（Rb+）密度，以确定天然脂质环境中BetP和EAAT 1中的K+配位。 为了研究利用转运蛋白的三聚体结构的动态调节脂质相互作用，我们将依赖于FTIR模式中的创新的基于扫描的光谱方法S-SNOM。

The secondary transporter function is highly affected by oligomerization. One prime example is the Na+ coupled betaine transporter BetP, a member of the BCCT family, which is regulated exclusively as a trimer. In trimeric state, specific lipid contacts as well as intra-trimeric interactions of terminal domains take place resulting in a re-orientation of individual protomers with respect to the membrane normal. As a consequence, the turn-over rate and cooperativity of Na+ coupling in BetP is altered in a K+ dependent manner. Being structurally unrelated, Na+/K+/H+-coupled glutamate transporters of the SLC1 family, e.g. EAAT1, share the trimeric assembly with BetP. The central ‘trimerization domain’ in SLC1 transporters deeply embedded in the membrane enables the characteristic and independent elevator movement of the three ‘transport domains’ during alternating access. In BetP, the C-terminal domain is the major transport regulator. Likewise, in EAAT1 and EAAT2, the respective C-terminal domains were shown to modify the glutamate uptake cycle and chloride permeability. In this project, we want to investigate if there are common mechanistic pattern in trimeric EAAT1 and BetP, by which regulatory and/or modulating lipid interactions affect the cooperativity of Na+ coupling and K+ antiport. Any mechanistic similarities between these evolutionary unrelated trimeric symporters might suggest very general principles in sodium-coupled transport regulation. Therefore, we want to perform a comparative structure-dynamics study between BetP and EAAT1. We want to investigate (1) the role of trimerization as interaction platform for lipids and modulators, and (2) the regulatory role of K+ on cooperative sodium coupling in BetP and EAAT1. To achieve this goal, we want to use these trimeric transporters, for which extensive structural information is already available to establish new tools in structural biology using mainly cryo scanning transmission electron microscopy (cryo-STEM) and single molecule scanning near-field optical microscopy (s-SNOM) in FTIR mode. By the means of cryo-STEM we want to assign K+ (Rb+) densities in cryo-EM structures to determine K+ coordination in BetP and EAAT1 in native lipid environment. To investigate dynamic regulatory lipid interactions exploiting the trimeric architecture of the transporters, we will rely on the innovative scanning-based spectroscopy method s-SNOM in an FTIR mode.