Structure and Function of Complexin

络合素的结构和功能

• 批准号: 9353435
• 负责人: David Eliezer
• 金额: $ 33.12万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353435
• 关键词: Affinity; Asses; Binding; Biological Assay; Brain; C-terminal; Communication; Dependence; Disease; Equilibrium; Exhibits; Exocytosis; Fluorescence; Hydrophobicity; In Vitro; Kinetics; Knowledge; Lipid Binding; Measurement; Measures; Mediating; Membrane; Mental disorders; Methods; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Nature; Nervous System Physiology; Neurons; Optics; Periodicity; Phospholipids; Play; Positioning Attribute; Process; Property; Protein Isoforms; Protein Region; Proteins; Regulation; Role; SNAP receptor; Spectrum Analysis; Structure; Synaptic Vesicles; Testing; Vesicle; base; design; farnesylation; fly; in vivo; mutant; nervous system disorder; neurotransmission; neurotransmitter release; presynaptic neurons; protein function; synaptic inhibition; vesicular release

Project Summary/Abstract: The protein complexin plays a critical role in the regulation of SNARE mediated synaptic vesicle exocytosis at presynaptic nerve terminals in the brain. Complexin functions both to inhibit spontaneous synaptic vesicle fusion and to enhance synchronized fusion. The central helix domain of complexin is required for both its inhibitory and its facilitatory functions, but additional regions of the protein, in particular the C- terminal domain (CTD), which follows the central helix and the accessory helix, which immediately precedes the central helix, are also required for complexin's full inhibitory function. Our objective is to understand the molecular basis for the functional roles of the CTD and the accessory helix. The CTD of complexin interacts with phospholipid membranes, and this interaction serves to localize complexin to synaptic vesicles in vivo and is required for complexin's inhibitory function. Two motifs within the CTD act to both localize complexin specifically to highly curved synaptic vesicle membranes and to activate complexin's inhibitory activity only when bound to synaptic vesicles, but important questions remain regarding the mechanisms by which membrane-binding contributes to complexin's inhibitory function. Specifically, the structural basis for the curvature-dependent interactions of the two CTD motifs with membranes remain unclear. In addition, many complexins feature a C-terminal CAAX box motif that leads to farnesylation, and the membrane-binding properties of farnesylated complexins have not been explored. This proposal aims at filling key gaps in our knowledge of how the structural features of membrane-bound complexin relate to the function of the protein. To do this, two specific aims will be pursued. (1) To characterize the structural basis for the membrane curvature-dependent structural transition of the amphipathic helix motif of complexin's CTD, to assess the role of membrane curvature dependent helix formation in regulating complexin's inhibitory function and to assess the influence of this transition on membrane binding affinity and kinetics. (2) To characterize the structural basis for membrane interactions by both farnesylated and unmodified C-terminal motifs in complexin's CTD, and to asses the functional implications of these interactions. The accessory helix of complexin also contributes significantly to the inhibition of neurotransmitter release. Recent results suggest that the accessory helix acts to nucleate and stabilize helical structure in the central helix, and thereby facilitates the interactions of the central helix with SNARE proteins. In a third specific aim (3) we will test this hypothesis by directly characterizing alterations in the helicity of the accessory and central helices, measuring how such alterations influence SNARE binding in vitro, and correlating these measurements with effects on complexin's inhibitory function in vivo. Together these three aims will serve to elucidate the molecular basis for the roles of complexin's CTD and accessory helix in the inhibition of neurotransmitter release.

项目摘要/摘要： 蛋白质复合蛋白在 SNARE 介导的突触小泡的调节中起着关键作用 大脑突触前神经末梢的胞吐作用。络合蛋白的作用是抑制自发性 突触小泡融合并增强同步融合。需要复合蛋白的中央螺旋结构域 因为它的抑制和促进功能，但蛋白质的其他区域，特别是 C- 末端结构域 (CTD)，位于中央螺旋和辅助螺旋之后，紧邻其前面 中央螺旋也是复合蛋白完全抑制功能所必需的。我们的目标是了解 CTD 和辅助螺旋功能作用的分子基础。 复合蛋白的 CTD 与磷脂膜相互作用，这种相互作用用于定位 复合蛋白在体内与突触小泡结合，并且是复合蛋白的抑制功能所必需的。里面有两个主题 CTD 的作用是将复合蛋白特异性地定位于高度弯曲的突触小泡膜并激活 复合蛋白仅在与突触小泡结合时才具​​有抑制活性，但仍然存在重要问题 膜结合促进络合蛋白抑制功能的机制。具体来说， 两个 CTD 基序与膜的曲率依赖性相互作用的结构基础仍然存在 不清楚。此外，许多复合物具有 C 端 CAAX 盒基序，可导致法尼基化，并且 法尼基化复合蛋白的膜结合特性尚未被探索。该提案旨在填补 我们对膜结合复合蛋白的结构特征与功能之间的关系的认识存在关键差距 的蛋白质。为此，将追求两个具体目标。 (1) 表征结构基础 复合蛋白 CTD 的两亲性螺旋基序的膜曲率依赖性结构转变， 评估膜曲率依赖性螺旋形成在调节复合素抑制功能中的作用 并评估这种转变对膜结合亲和力和动力学的影响。 (2) 表征 法尼基化和未修饰的 C 端基序的膜相互作用的结构基础 复合蛋白的 CTD，并评估这些相互作用的功能影响。 复合蛋白的副螺旋也对神经递质的抑制有显着贡献 发布。最近的结果表明，副螺旋的作用是使螺旋结构成核和稳定。 中央螺旋，从而促进中央螺旋与 SNARE 蛋白的相互作用。在第三个具体 目标 (3) 我们将通过直接表征附件螺旋度的变化来检验这一假设 中心螺旋，测量这些改变如何影响体外 SNARE 结合，并将这些相关联 测量对体内复合蛋白抑制功能的影响。 这三个目标将共同阐明复杂蛋白 CTD 作用的分子基础 和辅助螺旋抑制神经递质释放。