The structural dynamics of a glutamate transporter homologue

谷氨酸转运蛋白同源物的结构动力学

基本信息

批准号：
9322587
负责人：
Olga Boudker
金额：
$ 59.4万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-15 至 2018-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9322587
关键词：
Affect Anions Aspartate Ataxia Behavior Binding Binding Sites Biological Models Biophysics Brain Cations Cell membrane Chemicals Cognition Collaborations Computer Simulation Crystallization Crystallography Data Detergents Development Disease Electron Spin Resonance Spectroscopy Engineering Environment Event Excitatory Amino Acids Functional disorder Glutamate Transporter Glutamates Goals Homologous Gene Human Hydration status Individual Intervention Kinetics Knowledge Learning Light Lipid Bilayers Measurement Mediating Membrane Membrane Proteins Memory Methods Modeling Molecular Molecular Models Motion Movement Mutation Nature Neuroglia Neurons Neurotransmitters Pathologic Pathway interactions Peripheral Pharmacology Physiologic pulse Physiological Play Population Process Property Protein Analysis Protein Conformation Protein Dynamics Protein Engineering Proteins Publishing Research Role Sampling Signal Transduction Sodium Structure Synapses System Techniques Testing Thermodynamics Thick Time Transport Process X-Ray Crystallography base biophysical techniques clinically relevant computer studies excitotoxicity experimental study extracellular fluorescence imaging imaging modality mechanical properties molecular modeling monomer multidisciplinary mutant neurotransmitter uptake prevent public health relevance reaction rate response scaffold simulation single molecule single-molecule FRET symporter uptake

项目摘要

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 kinetic 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.

描述（由申请人提供）：在大脑中，谷氨酸转运蛋白居住在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，从而允许多个回合的信号传导和预防兴奋性。这些必需的蛋白质利用阳离子的电化学梯度的能量驱动神经递质的浓度摄取，还介导了被谷氨酸和钠结合所致的被动阴离子通量。尽管谷氨酸转运蛋白的功能障碍与多种疾病和病理状态有关，但药理学干预的策略至少部分是由于缺乏对其动力学机制的理解而受到很大的限制。为了填补这一空白，拟议的研究的总体目标是促进我们对谷氨酸转运蛋白的动态，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细菌同源物，GLTPH，这是一个完善的模型系统，事实证明，该系统在理解谷氨酸转运蛋白的结构和机制方面非常有用。使用该系统，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。谷氨酸转运蛋白的提议机制需要相对于脚手架结构域，该机制带有所谓的转运域的跨膜运动，该机制将底物横穿整个膜，相对于脚手架结构域，该结构域在膜上仍然很静态。我们的主要假设基于已发布的初步数据，是通过动态中间体发生的，其中传输域和支架之间的接口被部分水合，因此在结构上“解锁”。我们建议证实我们的假设，即这些“解锁”事件是运输周期的速率限制的，并且有利于动态状态的突变会加速运输。我们进一步试图检验我们的假设，即传输周期的类似“解锁”在校外或校外中间体介导阴离子的渗透。最后，我们旨在建立膜环境和电动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有关。在这个项目中，我们建立了一个基于广泛的协作，将晶体学和功能实验与单分子荧光成像方法相结合，使我们能够实时遵循域运动。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。总的来说，这些多学科的工作将确定可行性o开发变构转运蛋白激活剂，阐明生理相关阴离子渗透的机制，并评估膜的调节作用。