An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE ( Soluble N- Ethylmaleimidesensitive Factor Attachment Protein Receptor) Complex Assembly*
An Extended Helical Conformation in Domain 3a of Munc18-1 Provides a Template for SNARE ( Soluble N- Ethylmaleimidesensitive Factor Attachment Protein Receptor) Complex Assembly*
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DOI：
10.1074/jbc.m113.514273
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发表时间：
2014-04-04
影响因子：
4.8
通讯作者：
Soellner, Thomas H.
中科院分区：
文献类型：
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作者：
Parisotto, Daniel;Pfau, Maximilian;Soellner, Thomas H.
背景：Munc 18 -1是膜融合所必需的，但其潜在机制尚不清楚。结果：Munc 18 -1结构域3a的不同点突变对螺旋12的构象、VAMP 2结合和膜融合有不同的影响。结论：螺旋12的构象转换通过与VAMP 2的相互作用促进SNAREpin的组装。重要性：Munc 18 - 1是一种SEC 1/Munc 18蛋白，是突触传递中的关键调节蛋白，可促进或抑制SNARE复合物的组装。虽然Munc 18 -1和封闭的突触融合蛋白1之间的二元抑制相互作用已被很好地描述，但Munc 18 -1如何刺激膜融合的机制仍然难以捉摸。使用重建的测定，解决囊泡对接，引发，夹紧，和融合在突触胞吐，我们表明，Munc 18 -1的结构域3a中的螺旋12刺激SNAREpin组装和膜融合。螺旋12内的单点突变（L348 R）选择性地消除了VAMP 2结合和Munc 18 -1在膜融合中的刺激功能。相比之下，靶向螺旋12起始处的天然开关位点（P335 A）（其可导致延伸的螺旋构象）进一步加速脂质混合。结合结构建模，数据表明螺旋12为VAMP 2提供了折叠模板，加速了SNARE蛋白组装和膜融合。类似的SEC 1/Munc 18-SNARE相互作用在其他运输步骤可能提供了一个通用的机制，以驱动脂质双层合并。在神经元突触处，Munc 18 -1可以将对接的突触囊泡转化为容易释放的池。
Background: Munc18-1 is required for membrane fusion, but the underlying mechanism is unknown. Results: Distinct point mutations in domain 3a of Munc18-1 differentially affect the conformation of helix 12, VAMP2 binding, and membrane fusion. Conclusion: A conformational switch in helix 12 promotes SNAREpin assembly via the VAMP2 interaction. Significance: The Munc18-1-VAMP2 interaction may represent a general molecular mechanism of how SM proteins accelerate membrane fusion.Munc18-1, a SEC1/Munc18 protein and key regulatory protein in synaptic transmission, can either promote or inhibit SNARE complex assembly. Although the binary inhibitory interaction between Munc18-1 and closed syntaxin 1 is well described, the mechanism of how Munc18-1 stimulates membrane fusion remains elusive. Using a reconstituted assay that resolves vesicle docking, priming, clamping, and fusion during synaptic exocytosis, we show that helix 12 in domain 3a of Munc18-1 stimulates SNAREpin assembly and membrane fusion. A single point mutation (L348R) within helix 12 selectively abolishes VAMP2 binding and the stimulatory function of Munc18-1 in membrane fusion. In contrast, targeting a natural switch site (P335A) at the start of helix 12, which can result in an extended -helical conformation, further accelerates lipid-mixing. Together with structural modeling, the data suggest that helix 12 provides a folding template for VAMP2, accelerating SNAREpin assembly and membrane fusion. Analogous SEC1/Munc18-SNARE interactions at other transport steps may provide a general mechanism to drive lipid bilayer merger. At the neuronal synapse, Munc18-1 may convert docked synaptic vesicles into a readily releasable pool.