Structure of the synaptotagmin-SNARE interaction between membranes

膜间突触结合蛋白-SNARE 相互作用的结构

• 批准号: 10043788
• 负责人: Mike Fealey
• 金额: $ 6.49万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043788
• 关键词: Address; Amino Acids; Binding; Biological Assay; Biological Process; Biomedical Research; Biophysics; Brain; C2 Domain; Calcium; Calcium ion; Cell membrane; Charge; Chelating Agents; Complex; Cryoelectron Microscopy; Disease; Dissection; Docking; Elements; Environment; Event; Exocytosis; Goals; Heterogeneity; In Vitro; Investigation; Isotope Labeling; Isotopes; Lanthanoid Series Elements; Lead; Learning; Length; Link; Lipid Binding; Lipids; Measurement; Membrane; Membrane Fusion; Membrane Proteins; Memory; Methods; Modeling; Molecular; Molecular Conformation; Molecular Structure; Neurons; Neurosciences; Nuclear Magnetic Resonance; Penetration; Phosphorylation; Play; Positioning Attribute; Preparation; Process; Protein Isoforms; Protein Region; Proteins; Quantitative Reasoning; Research; Rest; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Series; Signal Transduction; Structure; Synapses; Synaptic Cleft; Synaptic Vesicles; Testing; Therapeutic; Training; Transmembrane Domain; VAMP-1; Validation; Vesicle; Work; career; chemical release; experimental study; fly; glycyl-glycyl-glycine; in vitro testing; in vivo; information processing; interdisciplinary approach; nanodisk; nervous system disorder; neurotransmission; neurotransmitter release; particle; programs; proteoliposomes; reconstitution; skills; stem; structural biology; synaptotagmin; synaptotagmin I; syntaxin 1; vesicle-associated membrane protein; vesicular release

The purpose of this proposal is to provide training that will prepare me for independent biomedical research focused on intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDRs) as well as the roles they play in regulating calcium-triggered synaptic vesicle exocytosis, a fundamental biological process that all neurons in the brain use to communicate. This will be accomplished by investigating the interactions between synaptotagmin-1 (Syt1), a protein that helps confer calcium-sensitivity to exocytosis and contains an IDR, and the SNARE proteins syntaxin-1, SNAP-25, and synaptobrevin, IDPs that assemble into a four-helix bundle that helps promote fusion of vesicle and plasma membranes in order to release vesicle contents into the synaptic cleft. Our understanding of how these proteins cooperate with one another to accomplish this goal has been limited by only studying their atomic structures in the absence of membrane bilayers, the most critical element of their natural cellular environment. This proposal aims to address this limitation by studying the molecular interaction of Syt1 with SNARE proteins while all are anchored to and actively bridging two separate membranes. First, in Aim 1 of this proposal, several nuclear magnetic resonance (NMR) methods will be used to determine the molecular structure of Syt1 by itself anchored to a lipid nanodisc by its transmembrane region in the absence of calcium ion. Importantly, this membrane-anchored Syt1 includes its long 60-amino acid IDR, which has not been stringently studied but may play a role in assisting the Syt1 interaction with SNAREs. This molecular structure will serve as a reference state for the protein at rest. Complementary to Aim 1, Aim 2 will combine NMR methods with cryo-electron microscopy (cryo-EM) to determine the molecular structure of Syt1 bound to the SNARE proteins as they actively bridge two nanodisc membranes, mimicking their arrangement just before a synaptic vesicle fuses with the plasma membrane. This molecular structure will serve as the primed reference state. Finally, the impact of calcium ion on this primed Syt1/trans-SNARE interaction will be determined using NMR and cryo-EM, revealing the molecular changes triggered by calcium that ultimately promote the membrane fusion of exocytosis. These results will significantly enhance our molecular understanding of how neurons release neurotransmitters and will provide an avenue for developing therapeutic strategies of the pre- synapse. Overall, execution of this proposal will equip me with several technical research skills and rigorous quantitative reasoning well suited for continued investigation of IDPs and IDRs and the roles they play in fundamental processes of molecular neuroscience.

这个提议的目的是为我提供培训，为我独立的生物医学研究做好准备，重点是内在无序蛋白（IDPs）和内在无序蛋白区域（IDRs），以及它们在调节钙触发的突触囊泡胞外作用中的作用，这是大脑中所有神经元用来交流的基本生物学过程。这将通过研究synaptotagmin-1 （Syt1）和SNARE蛋白syntaxin-1、SNAP-25和synaptobrevin之间的相互作用来完成。Syt1是一种蛋白质，有助于赋予钙对胞外分泌的敏感性，并含有IDR， syntaxin-1、SNAP-25和synaptobrevin是IDPs，它们组装成一个四螺旋束，有助于促进囊泡和质膜的融合，从而将囊泡内容物释放到突触间隙中。我们对这些蛋白质如何相互合作以实现这一目标的理解受到限制，因为我们只研究了它们在没有膜双层的情况下的原子结构，而膜双层是它们自然细胞环境中最关键的元素。本研究旨在通过研究Syt1与SNARE蛋白的分子相互作用来解决这一限制，而它们都被锚定在两个独立的膜上并主动桥接。首先，在本提案的目的1中，将使用几种核磁共振（NMR）方法来确定Syt1在缺乏钙离子的情况下通过其跨膜区域锚定在脂质纳米盘上的分子结构。重要的是，这种膜锚定的Syt1包括其长60个氨基酸的IDR，尚未被严格研究，但可能在协助Syt1与SNAREs相互作用中发挥作用。这种分子结构将作为静止蛋白质的参考状态。作为Aim 1的补充，Aim 2将结合核磁共振方法和低温电子显微镜（cryo-EM）来确定Syt1与SNARE蛋白结合的分子结构，因为它们主动桥接两个纳米盘膜，模拟它们在突触囊泡与质膜融合之前的排列。这个分子结构将作为引物参考态。最后，钙离子对Syt1/trans-SNARE相互作用的影响将通过核磁共振和低温电镜来确定，揭示钙触发的分子变化，最终促进胞外分泌的膜融合。这些结果将显著增强我们对神经元如何释放神经递质的分子理解，并将为开发突触前的治疗策略提供途径。总的来说，执行这个提案将使我具备一些技术研究技能和严格的定量推理能力，非常适合继续研究idp和idr及其在分子神经科学基本过程中的作用。