The structural dynamics of a glutamate transporter homologue

描述（由申请人提供）：在大脑中，谷氨酸转运蛋白位于神经胶质细胞和神经元的质膜中，位于谷氨酸能突触处，在那里它们介导神经递质摄取，允许多轮信号传导并防止兴奋性毒性。这些必需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摄取，并且还介导由谷氨酸和钠结合门控的被动阴离子通量。虽然谷氨酸转运体功能障碍与许多疾病和病理状态有关，但由于缺乏对它们的动力学机制的了解，药物干预策略受到很大限制。为了填补这一空白，拟议研究的总体目标是促进我们对谷氨酸转运蛋白的动力学，结构和功能之间关系的理解。在这些研究中，我们依赖于使用相关的细菌同源物GltPh，这是一种完善的模型系统，已证明其在理解谷氨酸转运蛋白的结构和机制方面非常有用。使用这个系统，我们建议建立限制底物摄取速率和关键中间体结构的事件的性质。所提出的谷氨酸转运蛋白的机制需要所谓的转运结构域的跨膜运动，其相对于支架结构域携带底物穿过膜，支架结构域在膜中基本上保持静止。我们的关键假设，根据已发表的初步数据，是这个过程发生通过动态中间体，其中的运输域和支架之间的界面变得部分水合，因此，结构上的“解锁”。我们建议证实我们的假设，这些“解锁”事件是速率限制的运输周期，有利于动态状态的突变加速运输。我们进一步试图测试我们的假设，类似的“解锁”的或关闭的途径中间体的运输周期介导阴离子渗透。最后，我们的目标是确定膜环境和通电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中的发现如何与真核谷氨酸转运蛋白相关。在这个项目中，我们已经建立了一个基础广泛的合作，结合晶体学和功能实验与单分子荧光成像方法，使我们能够在真实的时间内跟踪域运动;蛋白质工程;脉冲偶极电子自旋共振光谱;和多尺度计算和建模方法。总的来说，这些多学科的努力将建立开发变构转运蛋白激活剂的可行性，阐明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.