Molecular determinants of complexin clamping and activation function.
Molecular determinants of complexin clamping and activation function.
复制标题
络合蛋白夹紧和激活功能的分子决定因素。
DOI：
10.7554/elife.71938
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发表时间：
2022-04-20
期刊：
影响因子：
7.7
通讯作者：
Rothman, James E.
中科院分区：
文献类型：
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作者：
Bera, Manindra;Ramakrishnan, Sathish;Coleman, Jeff;Krishnakumar, Shyam S.;Rothman, James E.
先前我们报道了Synaptotagmin-1和Complexin协同地夹住SNARE组装过程以产生和维持停靠囊泡的池，所述停靠囊泡在Ca 2+流入时快速且同步地融合（Ramakrishnan等人，2020年）。在这里，使用相同的体外单囊泡融合试验，我们确定的复杂蛋白介导的融合钳的分子细节和它的作用，在Ca 2+激活。我们发现，延迟融合动力学，可能赋予突触结合蛋白-1，是需要复合蛋白阻断融合。系统的截短/突变分析表明，复杂蛋白的连续α-螺旋辅助中心结构域对其抑制功能是必不可少的，辅助螺旋与SNAREpin的特异性相互作用增强了这种功能。C-末端结构域通过与膜相互作用局部升高复合蛋白浓度来促进夹持。独立于它们的夹紧功能，复合蛋白的辅助中心螺旋结构域也有助于快速Ca 2+同步囊泡释放，通过增加从夹紧状态融合的可能性。
Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.