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

脂质对三聚体钠偶联同向转运体构象循环的作用

• 批准号: 442489199
• 负责人: Professorin Dr. Christine Maria Ziegler
• 金额: --
• 依托单位: Institut für Biophysik und physikalische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/442489199?language=en
• 关键词: Role; lipids; conformational; cycling; trimeric

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.

次级转运蛋白功能受寡聚的高度影响。一个典型的例子是BCCT家族的成员Na+耦合的Betaine Transporter betp，该转运蛋白betp仅被视为三聚体。在三聚体状态下，特定的脂质接触以及末端结构域的三传中的相互作用发生，导致单个药物相对于膜正常的重新定位。结果，BETP中Na+耦合的转换率和协调以K+依赖性方式改变。在结构无关的是SLC1家族的Na+/K+/H+偶联的谷氨酸转运蛋白，例如EAAT1，与Betp共享三聚合物组件。 SLC1转运蛋白中的中央“三聚域”深深嵌入膜中，可以在替代访问期间在三个“传输域”的特征和独立的基本运动。在BETP中，C末端结构域是主要的运输调节器。同样，在EAAT1和EAAT2中，相关的C末端结构域被证明可以改变谷氨酸摄取周期和氯化物的通透性。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械模式，通过该模式，调节和/或调节脂质相互作用会影响Na+耦合和K+ Antiport的配位。这些进化无关的三聚体分类器之间的任何机械相似性，这可能表明钠耦合转运调节中的非常一般的原理。因此，我们希望在BetP和EAAT1之间进行比较结构 - 动力学研究。我们想研究（1）修剪化作为脂质和调节剂的相互作用平台的作用，以及（2）K+在BetP和EAAT1中K+对合作钠耦合的调节作用。为了实现这一目标，我们希望使用这些三聚体转运蛋白，为此，已经可以使用主要的结构信息来建立结构生物学的新工具，其中主要是冷冻扫描传输电子显微镜（Cryo-STEM）和单分子扫描近场光学显微镜（S-SNOM）在FTIR模式下。通过冷冻词干的方式，我们希望在冷冻EM结构中分配K+（RB+）密度，以确定本机脂质环境中BetP和EAAT1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，我们将依靠基于创新的基于创新的扫描光谱法在FTIR模式下进行S-SNOM。