STRUCTURE OF A GLUTAMATE RECEPTOR BINDING DOMAIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 谷氨酸受体是脊椎动物脑内主要的兴奋性神经递质受体，参与多种正常和病理的神经功能。这些蛋白质通过在细胞外区域结合谷氨酸，并打开允许阳离子流入和流出神经元的内在离子通道来发挥作用。针对谷氨酸受体的药物可能在治疗癫痫、肌萎缩侧索硬化症和缺血性脑损伤等多种疾病方面具有相当大的潜力。我们正在研究两个重要的谷氨酸受体(GluR2和GluR3)，使用X射线结晶学和核磁共振和ESR光谱来了解其结构和动力学，并将结果与使用单通道记录(跨细胞膜离子电导测量)测量的蛋白质功能进行比较。结构工作是在蛋白质(GluR2 S1S2和GluR3 S1S2)的胞外配体结合域上进行的，这些蛋白质是由全长蛋白质衍生的可溶结构物。这些蛋白质具有双叶结构，谷氨酸及其衍生物的结合位置在两个叶之间的界面上。对于GluR2，以前的工作表明，结合配体时叶关闭的程度可能与蛋白质的功能有关。我们对核磁共振波谱和最近的晶体结构的初步工作表明，结构和功能之间的关系可能更复杂，涉及蛋白质的灵活性。在揭示可能运动范围的条件下获得额外的结构，对于理解激动剂结合的功能后果是必不可少的。与Sondermann实验室合作，我们获得了GluR2 S1S2与几个新配体结合的结构(分辨率为1.5到1.7埃)。本周期所需的时间将用于确定在两种结晶条件下带有三个新配体的GluR2 S1S2的结构，我们怀疑这将提供蛋白质可能运动范围的指示。我们也有GluR3 S1S2的晶体，其结构尚未确定。这种蛋白质也可与三种不同的配体结合。除了了解其在中枢神经系统中的关键功能的重要性外，将这些研究扩展到GluR3亚型的原因是我们对GluR3的功能研究近年来有了很大的进步，希望详细了解结合区域的结构变化与离子通过细胞膜的电导之间的联系是非常有希望的。我们已经确定了条件，并生长了所有用于这些研究的晶体。如上所述，我们从GluR2获得的晶体衍射到高分辨率(1.5到1.7埃)，我们预计我们获得的新晶体也会得到类似的结果。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glutamate receptors are the major excitatory neurotransmitter receptors in vertebrate brain and are involved in a variety of normal and pathological neuronal functions. These proteins function by binding glutamate in an extracellular domain and opening an intrinsic ion channel that allows cations to flow in and out of the neuron. Drugs targeted to glutamate receptors may have considerable potential for treating such diverse disorders as epilepsy, amyotrophic lateral sclerosis, and ischemic brain damage. We are studying two important glutamate receptors (GluR2 and GluR3), using X-ray crystallography and NMR and ESR spectroscopy to understand the structure and dynamics and to compare the results with the function of the protein measured using single channel recording (measurement of ion conductance across the cell membrane). The structural work is done on the extracellular ligand-binding domains of the proteins (GluR2 S1S2 and GluR3 S1S2), which are a soluble constructs derived from the full-length proteins. The proteins have a bilobed structure with the binding site for glutamate and derivatives at the interface between the two lobes. For GluR2, previous work has suggested that the degree to which the lobes close upon binding of ligand may relate to the function of the protein. Our initial work with NMR spectroscopy and our recent crystal structures, suggest that the relationship between structure and function may be more complicated, involving protein flexibility. Obtaining additional structures, under conditions that reveal the range of possible motions, is essential for understanding the functional consequences of agonist binding. In collaboration with the Sondermann laboratory, we have obtained the structures of GluR2 S1S2 bound to several new ligands (1.5 to 1.7 angstrom resolution). The time requested in this cycle will be used to determine structures of GluR2 S1S2 with three new ligands under two crystallization conditions that we suspect will provide an indication of the range of possible motions of the protein. We also have crystals for GluR3 S1S2, the structure of which has not yet been determined. This protein is also available bound to at three different ligands. In addition the importance of understanding its crucial function in the central nervous system, the rationale for expanding these studies to the GluR3 subtype is that our functional studies of GluR3 have advanced considerably in recent years, and the hope of understanding in detail the link between structural changes in the binding domain to the conductance of ions through the cell membrane is very promising with this subtype. We have determined the conditions and have grown all of the crystals to be used in these studies. As noted above, the crystals that we have obtained from GluR2 diffract to high resolution (1.5 to 1.7 angstroms), and we anticipate similar results from the new crystals that we have obtained.