The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission
The Interaction of Munc18-1 Helix 11 and 12 with the Central Region of the VAMP2 SNARE Motif Is Essential for SNARE Templating and Synaptic Transmission
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DOI：
10.1523/eneuro.0278-20.2020
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发表时间：
2020-11-01
期刊：
影响因子：
3.4
通讯作者：
Soellner, Thomas H.
中科院分区：
文献类型：
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作者：
Andre, Timon;Classen, Jessica;Soellner, Thomas H.
Sec 1/Munc 18蛋白在启动N-乙基马来酰亚胺敏感因子附着蛋白受体（SNARE）复合物（分子融合机制）的组装中发挥关键作用。采用比较结构建模，位点特异性交联与光活化的非天然氨基酸对苯甲酰基-苯丙氨酸（Bpa）和重组囊泡对接/融合试验的单氨基酸取代，我们映射Munc 18 -1和神经元的v-SNARE VAMP 2与单氨基酸分辨率之间的结合界面。我们的研究结果表明，Munc 18 -1中结构域3a的螺旋11和12与覆盖层-4至+5区域的VAMP 2 SNARE基序相互作用。螺旋11中的残基Q301在VAMP 2结合和模板复合物形成中起关键作用。VAMP 2结合缺陷突变体Munc 18 -1 Q301 D在重构融合测定中不刺激脂质混合。神经元陷阱组织者Munc 13 -1也与VAMP 2结合，但不能绕过Munc 18 -1.VAMP2相互作用的要求。重要的是，Munc 18 -1缺陷神经元中的Munc 18 -1 Q301 D表达严重减少突触传递，证明了Munc 18 -1.VAMP2相互作用的生理学意义。
Sec1/Munc18 proteins play a key role in initiating the assembly of N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes, the molecular fusion machinery. Employing comparative structure modeling, site specific crosslinking by single amino acid substitutions with the photoactivatable unnatural amino acid p-Benzoyl-phenylalanine (Bpa) and reconstituted vesicle docking/fusion assays, we mapped the binding interface between Munc18-1 and the neuronal v-SNARE VAMP2 with single amino acid resolution. Our results show that helices 11 and 12 of domain 3a in Munc18-1 interact with the VAMP2 SNARE motif covering the region from layers -4 to +5. Residue Q301 in helix 11 plays a pivotal role in VAMP2 binding and template complex formation. A VAMP2 binding deficient mutant, Munc18-1 Q301D, does not stimulate lipid mixing in a reconstituted fusion assay. The neuronal SNARE-organizer Munc13-1, which also binds VAMP2, does not bypass the requirement for the Munc18-1.VAMP2 interaction. Importantly, Munc18-1 Q301D expression in Munc18-1 deficient neurons severely reduces synaptic transmission, demonstrating the physiological significance of the Munc18-1.VAMP2 interaction.