The structural dynamics of a glutamate transporter homologue

描述(申请人提供)：在大脑中，谷氨酸转运体位于谷氨酸能突触的神经胶质细胞和神经元的质膜中，在那里它们介导神经递质的摄取，允许多轮信号传递并防止兴奋性毒性。这些必需的蛋白质利用阳离子的电化学梯度的能量来驱动神经递质的集中吸收，并通过谷氨酸和钠结合来调节被动阴离子通量。虽然谷氨酸转运体功能障碍与多种疾病和病理状态有关，但药物干预的策略非常有限，至少部分原因是由于缺乏对其KinETC机制的了解。为了填补这一空白，拟议中的研究的首要目标是促进我们对谷氨酸转运体的动力学、结构和功能之间的关系的理解。在这些研究中，我们依赖于一种相关的细菌同源物GltPh的使用，这是一个成熟的模型系统，已被证明在理解谷氨酸转运蛋白的结构和机制方面非常有用。利用这个系统，我们建议确定限制底物吸收速率的事件的性质和关键中间体的结构。所提出的谷氨酸转运体的机制需要所谓的转运域相对于支架结构域的跨膜移动，转运域携带底物穿过膜，支架结构域基本上保持在膜上不动。根据已公布的初步数据，我们的关键假设是，这一过程通过动态中间体发生，其中运输结构域和支架之间的界面变得部分水合，因此在结构上是“解锁的”。我们建议证实我们的假设，即这些“解锁”事件是对运输周期的速率限制，并且有利于动态的突变加速运输。我们进一步试图测试我们的假设，即类似的“解锁”的途径上或途径外的运输周期中介阴离子渗透。最后，我们的目标是确定膜环境和通电的跨膜电化学梯度如何影响转运蛋白的动力学和热力学，以及我们在GltPh上的发现如何与真核细胞的谷氨酸转运蛋白相关。在这个项目中，我们建立了广泛的合作，将结晶学和功能实验与单分子荧光成像方法相结合，使我们能够实时跟踪结构域的运动；蛋白质工程；脉冲偶极电子自旋共振光谱；以及多尺度计算和建模方法。总而言之，这些多学科的努力将建立开发变构转运体激活剂的可行性，阐明与生理相关的阴离子渗透机制，并评估膜的调节作用。

DESCRIPTION (provided by applicant): In the brain, glutamate transporters reside in the plasma membranes of glial cells and neurons at glutamatergic synapses, where they mediate neurotransmitter uptake, allowing multiple rounds of signaling and preventing excitotoxicity. These essential proteins harness the energy of the electrochemical gradients of cations to drive concentrative uptake of the neurotransmitter, and also mediate passive anion fluxes gated by glutamate and sodium binding. Although dysfunction of glutamate transporters is associated with a plethora of diseases and pathological states, strategies for the pharmacological intervention are greatly limited due, at least in part, to the lack of understanding of their kinetc mechanism. To fill this gap, the overarching goal of the proposed research is to advance our understanding of the relationship between the dynamics, structure and function of glutamate transporters. In these studies we rely on the use of a related bacterial homologue, GltPh, a well- established model system, which has proven exceptionally useful in understanding the structure and mechanism of glutamate transporters. Using this system, we propose to establish the nature of the events that limit the rates of substrate uptake and the structures of the key intermediates. The proposed mechanism of glutamate transporters entails trans-membrane movements of the so-called transport domain, which carries the substrate across the membrane, relative to the scaffolding domain, which remains largely static in the membrane. Our key postulate, based on the published preliminary data, is that this process occurs via dynamic intermediates, in which the interface between the transport domain and the scaffold becomes partially hydrated and, therefore, structurally "unlocked". We propose to substantiate our hypothesis that these "unlocking" events are rate limiting to the transport cycle, and that mutations that favor the dynamic states accelerate transport. We further seek to test our hypothesis that similar "unlocked" on- or off-pathway intermediates of the transport cycle mediate anion permeation. Finally, we aim to establish how the membrane environment and the energizing trans-membrane electrochemical gradients affect the dynamics and the thermodynamics of the transporter and how our findings in GltPh relate to the eukaryotic glutamate transporters. In this project, we have established a broad-based collaboration, combining crystallography and functional experiments with single molecule fluorescence imaging methods that allow us to follow domain movements in real time; protein engineering; pulsed dipolar electron spin resonance spectroscopy; and multi-scale computational and modeling approaches. Collectively, these multidisciplinary efforts will establish the feasibility o developing allosteric transporter activators, shed light on the mechanism of physiologically relevant anion permeation, and evaluate the modulatory role of the membrane.