Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.
Interaction of the Complexin Accessory Helix with Synaptobrevin Regulates Spontaneous Fusion.
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DOI：
10.1016/j.bpj.2016.09.017
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发表时间：
2016-11
影响因子：
3.4
通讯作者：
Alexander Vasin;Dina Volfson;J. Littleton;M. Bykhovskaia
中科院分区：
文献类型：
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作者：
Alexander Vasin;Dina Volfson;J. Littleton;M. Bykhovskaia
神经元递质通过突触小泡与质膜的融合从神经末梢释放。囊泡通过由膜附着 (t-SNARE) 和囊泡附着 (v-SNARE) 蛋白质组成的特殊蛋白质机制附着在膜上，这些蛋白质拉链在一起形成卷曲螺旋 SNARE 束，使两个融合膜紧密相连。神经递质释放可以响应动作电位或通过自发融合发生。胞质蛋白 Complexin (Cpx) 与 SNARE 复合体结合，并通过充当融合钳来限制自发的胞吐作用。我们之前提出了一个模型，其中 Cpx 和 v-SNARE 之间的相互作用充当弹簧，以防止 SNARE 复合体过早拉开，从而降低融合的可能性。为了测试这个模型，我们结合了分子动力学 (MD) 模拟和果蝇 Cpx 和 SNARE 的定点诱变。果蝇Cpx-SNARE复合体的MD模拟表明，Cpx与v-SNARE的相互作用促进了v-SNARE从核心SNARE束中解开。我们研究了 thesyx3-69paralytic 突变体的夹紧特性，该突变体在 t-SNARE 中具有单点突变，并表现出增强的自发释放。 MD 模拟表明 Cpx 与 SNARE 束的相互作用发生了改变，这阻碍了 Cpx 对 v-SNARE 的解开，从而影响了夹紧。我们使用我们的模型来预测突变，这些突变应该增强 Cpx 阻止 SNARE 复合体完全组装的能力。 MD 模拟预测，Cpx 辅助螺旋和 v-SNARE 之间的相互作用减弱将增强 Cpx 的灵活性，从而促进 SNARE 的分离，减少自发融合。我们生成了转基因果蝇，其 Cpx 和 v-SNARE 突变破坏了这两种蛋白质之间的盐桥。正如预测的那样，两条线都表现出对自发释放的选择性抑制，表明 Cpx 充当限制完全 SNARE 拉链的融合夹。
Neuronal transmitters are released from nerve terminals via the fusion of synaptic vesicles with the plasma membrane. Vesicles attach to membranes via a specialized protein machinery composed of membrane-attached (t-SNARE) and vesicle-attached (v-SNARE) proteins that zipper together to form a coiled-coil SNARE bundle that brings the two fusing membranes into close proximity. Neurotransmitter release may occur either in response to an action potential or through spontaneous fusion. A cytosolic protein, Complexin (Cpx), binds the SNARE complex and restricts spontaneous exocytosis by acting as a fusion clamp. We previously proposed a model in which the interaction between Cpx and the v-SNARE serves as a spring to prevent premature zippering of the SNARE complex, thereby reducing the likelihood of fusion. To test this model, we combined molecular-dynamics (MD) simulations and site-directed mutagenesis of Cpx and SNAREs inDrosophila. MD simulations of theDrosophilaCpx-SNARE complex demonstrated that Cpx's interaction with the v-SNARE promotes unraveling of the v-SNARE off the core SNARE bundle. We investigated clamping properties in thesyx3-69paralytic mutant, which has a single-point mutation in the t-SNARE and displays enhanced spontaneous release. MD simulations demonstrated an altered interaction of Cpx with the SNARE bundle that hindered v-SNARE unraveling by Cpx, thus compromising clamping. We used our model to predict mutations that should enhance the ability of Cpx to prevent full assembly of the SNARE complex. MD simulations predicted that a weakened interaction between the Cpx accessory helix and the v-SNARE would enhance Cpx flexibility and thus promote separation of SNAREs, reducing spontaneous fusion. We generated transgenicDrosophilawith mutations in Cpx and the v-SNARE that disrupted a salt bridge between these two proteins. As predicted, both lines demonstrated a selective inhibition in spontaneous release, suggesting that Cpx acts as a fusion clamp that restricts full SNARE zippering.