Assembly, structure, and function of activated SNARE complexes and their regulation by NSF/αSNAP, Munc18, Munc13, complexin, and synaptotagmin

项目总结 本项目的总体目标是阐明该基因的精确分子机制和调控。 驱动胞吐以控制神经末梢神经递质释放的融合机。这个 位于突触小泡和突触前质膜中的SNARE分子的组装 中心舞台，并为这一过程提供动力。即使我们完全知道 在原子细节上融合后组装的顺式圈套复合体，并具有详细的构象模型 在融合之前的几个陷阱，我们不确切地知道(I)它们是如何调节的 Munc18和Munc13等蛋白质在质膜上形成活性受体复合体，(Ii)如何 这种受体陷阱复合体在相遇时与突触小泡陷阱接合，以及(Iii)这是如何 高能反式SNARE复合体最终由突触小泡蛋白synaptopagmin和 钙进行充分的组装和融合。 由三位专家领导领导的三个项目，涉及神经元的生物化学、结构生物学和生物物理学 胞外膜融合的设计是为了联合解开驱动细胞 神经元融合机通过囊泡对接、引爆和融合的步骤，以尽可能高的速度 结构和时间分辨率。该团队将寻求定义Active的结构和配置 突触前受体SNARE复合体和融合限制性反式SNARE复合体 膜，研究小组将努力揭示钙-突触凝集素 与膜和/或复合体接触以释放它们的融合限制。 为了实现这一目标，团队将使用独特的方法组合，范围从高度创新 重建相关蛋白质的生化程序，EPR，鹿，和核磁共振波谱 表征膜环境中的相关结构，以及FLIC和单泡TIRF显微镜 测量膜的拓扑结构，并在毫秒级上读出融合。

PROJECT SUMMARY The overall goal of this program project is to elucidate the precise molecular mechanism and regulation of the fusion machine that drives exocytosis for the controlled release of neurotransmitter at nerve terminals. The assembly of SNARE molecules residing in the synaptic vesicle and presynaptic plasma membrane takes center stage and provides the driving energy for this process. Even though we know the structure of the fully assembled cis-SNARE complex after fusion in atomic detail and have detailed conformational models for several of the SNAREs before fusion, we do not precisely know how (i) they are conditioned with regulatory proteins such as Munc18 and Munc13 to form an active acceptor complex on the plasma membrane, (ii) how this acceptor SNARE complex engages with the synaptic vesicle SNARE upon encounter, and (iii) how this high-energy trans-SNARE complex is ultimately triggered by the synaptic vesicle protein synaptotagmin and calcium to proceed to full assembly and fusion. Three projects led by three expert leaders in the biochemistry, structural biology, and biophysics of neuronal exocytotic membrane fusion are designed to jointly unravel the precise molecular interactions that drive the neuronal fusion machine through the vesicle docking, priming, and fusion steps with the highest possible structural and time resolution. The team will seek to define the structures and configurations of the active presynaptic acceptor SNARE complex and the fusion-restricted trans-SNARE complex between two membranes, and the team will strive to uncover the molecular mechanism, by which calcium-synaptotagmin engages with the membranes and/or complex to release their fusion-restriction. To achieve this goal the team will use a unique combination of approaches ranging from highly innovative biochemical procedures to reconstitute the relevant proteins, EPR, DEER, and NMR spectroscopy to characterize the pertinent structures in membrane environments, and FLIC and single vesicle TIRF microscopy to measure membrane topology and read out fusion on the millisecond timescale.