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。一个典型的例子是Na+偶联甜菜碱转运蛋白BetP，它是BCCT家族的一员，仅作为三聚体进行调节。在三聚体状态下，发生特定的脂质接触以及末端结构域的三聚体内部相互作用，导致单个原聚体相对于膜正常蛋白的重新定向。因此，BetP中Na+偶联的周转率和协同性以K+依赖的方式改变。SLC1家族的Na+/K+/H+偶联谷氨酸转运体（如EAAT1）与BetP共享三聚体组装体，但它们在结构上不相关。SLC1转运体的中心“三聚结构域”深深嵌入膜中，使三个“转运结构域”在交替进入时具有特征和独立的升降机运动。在BetP中，c端结构域是主要的转运调节因子。同样，在EAAT1和EAAT2中，各自的c端结构域被证明可以改变谷氨酸摄取周期和氯离子通透性。在这个项目中，我们想研究三聚体EAAT1和BetP中是否存在共同的机制模式，通过这种机制，调节和/或调节脂质相互作用影响Na+偶联和K+反端口的协同性。在这些进化上不相关的三聚体同源基因之间的任何机制上的相似性可能暗示了钠偶联运输调控的非常普遍的原则。因此，我们希望对BetP和EAAT1进行结构动力学比较研究。我们希望研究(1)三聚化作为脂质和调节剂相互作用平台的作用，以及(2)K+对BetP和EAAT1中协同钠偶联的调节作用。为了实现这一目标，我们希望利用这些三聚体转运体，其中大量的结构信息已经可用来建立结构生物学的新工具，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，我们希望在cryo-EM结构中分配K+ (Rb+)密度，以确定天然脂质环境中BetP和EAAT1中K+的配位。为了研究利用转运体三聚体结构的动态调节脂质相互作用，我们将在FTIR模式下依靠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.