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.

项目概要/摘要： 复合蛋白在SNARE介导的突触囊泡的调控中起着关键作用 突触前神经末梢的胞吐作用。复合蛋白的功能是抑制自发的 突触囊泡融合和增强同步融合。复合素的中心螺旋结构域是必需的 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），其跟随中心螺旋和辅助螺旋，其紧接在 中心螺旋也是复合蛋白的全部抑制功能所必需的。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 在体内，复合蛋白与突触囊泡结合，并且是复合蛋白的抑制功能所必需的。两个主题在 CTD既可以将复合蛋白特异性地定位于高度弯曲的突触囊泡膜， 复合蛋白的抑制活性，只有当结合到突触囊泡，但重要的问题仍然存在， 膜结合有助于复合蛋白抑制功能的机制。具体而言是 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，许多复合蛋白具有导致法尼基化的C-末端CAAX盒基序，并且其功能是通过在C-末端CAAX盒基序中引入法尼基化来实现的。 法尼基化复合蛋白的膜结合性质尚未被探索。该提案旨在填补 我们对膜结合复合蛋白的结构特征如何与功能相关的知识中的关键空白 蛋白质。为此，将追求两个具体目标。(1)为了描述 膜曲率依赖的结构转换的两亲性螺旋基序的复合蛋白的CTD， 评估膜曲率依赖性螺旋形成在调节复合蛋白抑制功能中的作用 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-末端基序与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意义。 复合蛋白的辅助螺旋也对神经递质的抑制有重要作用 release.最近的研究结果表明，辅助螺旋的作用是成核和稳定的螺旋结构中， 中心螺旋，从而促进中心螺旋与SNARE蛋白的相互作用。在第三个具体 目的（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，测量这种改变如何影响SNARE体外结合，并将这些 测定了复合素在体内的抑制功能。 这三个目标将有助于阐明复合蛋白CTD作用的分子基础 和辅助螺旋在抑制神经递质释放中的作用。