Conformation and Membrane Position of the Region Linking the Two C2 Domains in Synaptotagmin 1 by Site-Directed Spin Labeling
Conformation and Membrane Position of the Region Linking the Two C2 Domains in Synaptotagmin 1 by Site-Directed Spin Labeling
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DOI：
10.1021/bi801470m
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发表时间：
2008-11-25
期刊：
影响因子：
2.9
通讯作者：
Cafiso, David S.
中科院分区：
文献类型：
--
作者：
Huang, Hao;Cafiso, David S.
突触结合蛋白1（Synaptotagmin 1，syt 1）是一种位于突触囊泡上的膜蛋白，是神经元胞吐的钙离子传感器。突触结合蛋白1含有两个C2结构域，C2 A和C2B，其结合Ca 2+离子、膜和SNARE。在此，使用定点自旋标记（SDSL）来确定在包含两个C2结构域（syt 1C 2AB）的水溶性构建体中连接两个C2结构域的区域的位置和动力学。从这个区域的EPR线形状的分析表明，连接器是灵活的和非结构化时，sytl是在溶液中或结合到脂质双层。纳秒动力学的连接器不改变，在存在或不存在的Ca 2+，这表明有没有Ca 2+依赖的两个域之间的分子内关联。当syt 1C 2AB是膜结合的，连接器相对于膜界面的位置通过测量自旋标记的syt 1C 2AB突变体与可溶性和膜结合的Ni（II）螯合物的碰撞参数来确定。这些数据表明，接头不穿透膜表面，但位于离双层表面约7-10埃处。此外，当syt 1C 2AB与SNARE复合物结合时，接头保持柔性，表明该接头与SNARE之间的直接相互作用不介导缔合。这些数据表明，两个C2结构域的syt 1相互作用独立的膜界面上，或当绑定到SNARES。
Synaptotagmin 1 (syt1) is an integral membrane protein localized on the synaptic vesicle that acts as the Ca2+ sensor for neuronal exocytosis. Synaptotagmin 1 contains two C2 domains, C2A and C2B, which bind Ca2+ ions, membranes, and SNAREs. Here, site-directed spin labeling (SDSL) was used to determine the position and dynamics of the region that links the two C2 domains in a water soluble construct encompassing the two C2 domains (syt1C2AB). An analysis of the EPR line shapes from this region indicates that the linker is flexible and unstructured when sytl is in solution or bound to lipid bilayers. The nanosecond dynamics of the linker does not change, in the presence or absence of Ca2+, suggesting that there is no Ca2+-dependent intramolecular association between the two domains. When syt1C2AB is membrane-bound, the position of the linker relative to the membrane interface was determined by measuring parameters for the collision of the spin-labeled syt1C2AB mutants with both soluble and membrane-bound Ni(II) chelates. These data indicate that the linker does not penetrate the membrane surface but lies approximately 7-10 angstrom from the bilayer surface. In addition, the linker remains flexible when syt1C2AB binds to the SNARE complex, indicating that direct interactions between this linker and the SNAREs do not mediate association. These data suggest that the two C2 domains of syt1 interact independently on the membrane interface, or when bound to SNARES.