Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States.
Elife. 2020 Aug 18;9:e57154. doi: 10.7554/eLife.57154.
The Ca sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving 'polybasic', 'primary' and 'tripartite' interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca-free synaptotagmin-1 binds to SNARE complexes anchored on PIP-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca induces tight binding of synaptotagmin-1 to PIP-containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca-dependent synaptotagmin-1-PIP-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca releases this interaction, inducing PIP/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.
钙离子传感器突触融合蛋白-1 和 SNARE 复合物共同作用触发神经递质释放。结构研究阐明了突触融合蛋白-1 与 SNARE 复合物的三种不同结合模式，涉及突触融合蛋白-1 的“多碱性”、“主要”和“三分体”界面。我们使用 NMR 和荧光光谱学研究了这些相互作用。在溶液中，突触融合蛋白-1 通过多碱性和主要界面与 SNARE 复合物结合。在含有 PIP 的纳米盘上锚定的 SNARE 复合物上，无 Ca 时的突触融合蛋白-1 结合。位于主要界面的 R398Q/R399Q 和 E295A/Y338W 突变强烈损害神经递质释放，分别破坏和增强突触融合蛋白-1-SNARE 复合物的结合。Ca 诱导突触融合蛋白-1 与含有 PIP 的纳米盘紧密结合，破坏突触融合蛋白-1-SNARE 相互作用。多碱性区突变对 Ca 依赖性突触融合蛋白-1-PIP-膜相互作用的特定影响与其对释放的影响相关。我们的数据表明，突触融合蛋白-1 通过主要界面与 SNARE 复合物结合，而 Ca 释放这种相互作用，诱导 PIP/膜结合，并允许突触融合蛋白-1 和 SNARE 之间在膜融合中合作，触发释放。
