The Neuronal Priming Complex

描述（由申请人提供）：尽管我们在理解神经元中Ca+2依赖性胞吐作用方面取得了很大进展，但仍然没有清楚地理解Ca+2和胞吐作用之间的关系。对这一过程的准确看法是理解大脑功能的基础，对未来神经系统疾病的治疗至关重要。N型Ca+2通道与胞吐蛋白syntaxin和SNAP-25相互作用;然而，这些相互作用的结构基础仍然不清楚。通过详细的一级和二级结构分析，我们发现N型Ca+2通道具有SNARE样基序，该基序可能介导其与syntaxin和SNAP-25的相互作用。本R21项目的目的1将测试以下假设：N型Ca+2通道充当Q-SNARE，并且与突触融合蛋白和SNAP-25的相互作用遵循在顺式SNARE中观察到的一些相同结构模式。这种Ca+2通道“陷阱”不涉及突触融合蛋白-SNAP-25.synaptobrevin复合物的融合活性，但可能有助于将这些组分定位于胞吐作用前的Ca+2通道。利用重组SNARE蛋白的独特性质，我们期望表明N型Ca+2通道的synprint结构域与syntaxin和SNAP-25以与其他SNARE蛋白类似的方式相互作用。目的2为进一步研究胞吐过程中的Ca+2感受器--人突触结合蛋白1 C2 A-C2B的结构奠定基础。这种蛋白质还与Ca+2通道、突触融合蛋白和SNAP-25在多蛋白复合物中相互作用。作为最终多蛋白分析的前奏，我们将使用各种创新的结晶学技术来完善突触结合蛋白的结晶条件，这些技术旨在利用突触结合蛋白独特的脂质缔合特性。一旦完成，这两个目标将为我们提供工具和理解，以进一步确定这种蛋白质在Ca+2依赖性胞吐中的作用。在未来的R 01资助下，我们将能够将这项工作扩展到对这种独特的SNARE复合物进行更广泛的生物物理学和晶体学研究，并完成人类突触结合蛋白1 C2 A-C2B的高分辨率晶体结构。

DESCRIPTION (provided by applicant): Although we have made great strides in understanding Ca+2-dependent exocytosis in the neuron, there is still not a clear understanding of the relationship between Ca+2 and exocytosis. An accurate view of this process is fundamental to understanding brain function, and crucial to the future treatment of neurological diseases. The N-type Ca+2 channel interacts with the exocytosis proteins syntaxin and SNAP-25; however, the structural basis of these interactions is still not clear. Through detailed primary and secondary structural analysis, we found that the N-type Ca+2 channel possesses a SNARE-like motif that may mediate its interactions with syntaxin and SNAP-25. Aim 1 of this R21 project will test the hypothesis that the N-type Ca+2 channel acts as a Q-SNARE, and that the interactions with syntaxin and SNAP-25 follow some of the same structural patterns observed in the cis-SNARE. This Ca+2 channel "SNARE" is not implicated in the fusogenic activities of the syntaxin-SNAP-25.synaptobrevin complex, but may serve to localize these components to the Ca+2 channel immediately before exocytosis. Taking advantage of the unique properties of the recombinant SNARE proteins, we expect to show that the synprint domain of the N-type Ca+2 channel interacts with syntaxin and SNAP-25 in a similar manner as do other SNARE proteins. Aim 2 will lay the foundation for further structural work on human synaptotagmin 1 C2A-C2B, the Ca+2 sensor in exocytosis. This protein also interacts with the Ca+2 channel, syntaxin and SNAP-25 in a multiprotein complex. As a prelude to eventual multiprotein analysis, we will refine the crystallization conditions of synaptotagmin using a variety of innovative crystallographic techniques designed to take advantage of synaptotagmin's unique lipid-associating properties. Once completed, these two aims will provide us with the tools and understanding to further define this protein's role in Ca+2-dependent exocytosis. Under future R01 funding we will be able to extend this work to more extensive biophysical and crystallographic studies of this unique SNARE complex, and complete the high-resolution crystal structure of human synaptotagmin 1 C2A-C2B.