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

二次转运蛋白功能受齐聚作用的影响很大。一个最好的例子是钠偶联甜菜碱转运体BetP，它是BCCT家族的成员，只作为三聚体受到调控。在三聚体状态下，发生特定的脂类接触以及末端结构域的三聚体内相互作用，导致单个原型相对于膜正常方向的重新定位。结果表明，Na偶联在BetP中的转化率和协作性随K的变化而变化。由于结构上不相关，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。SLc1转运体的中心“三聚化结构域”深深嵌入在膜中，使得三个“转运域”在交替进入过程中能够进行独特而独立的电梯运动。在BetP中，C-末端结构域是主要的运输调节因子。同样，在EAAT1和EAAT2中，各自的C-末端结构域被证明改变了谷氨酸摄取周期和氯的通透性。在这个项目中，我们想要研究在三聚体EAAT1和BetP中是否存在共同的机制模式，通过这些机制来调节和/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转运体之间的任何机制上的相似之处都可能暗示着钠偶联转运调节的非常普遍的原则。因此，我们希望对BetP和EAAT1进行结构动力学比较研究。我们想要研究(1)三聚化作为脂类和调节剂的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，我们希望利用这些已经获得广泛结构信息的三聚体转运体来建立结构生物学的新工具，主要使用低温扫描电子显微镜(CRYO-STEM)和单分子扫描近场光学显微镜(S-SNOM)。通过低温扫描电子显微镜，我们对低温EM结构中的K(Rb)密度进行了归属，以确定在天然脂环境中BetP和EAAT1中的K配位。为了利用转运蛋白的三聚体结构来研究动态调节脂质相互作用，我们将依赖于创新的基于扫描的光谱方法，即傅立叶变换红外光谱模式下的S-近红外光谱分析。

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.