Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

中文翻译：

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突触前Munc13-1的Trp-588在配体结合和膜移位中的关键作用。

Munc13-1是突触前活性区蛋白，对于神经递质的释放和大脑中突触前可塑性至关重要。该多结构域支架蛋白包含与激活剂二酰基甘油/佛波醇酯结合的C1域。尽管C1结构域与蛋白激酶C（PKC）的C1结构域具有紧密的结构同源性，但22位（全长Munc13-1中的588）处的色氨酸残基遮盖了激活剂结合袋，而PKC并非如此。为了阐明该色氨酸的作用，我们在全长Munc13-1中产生了W22A，W22K，W22D，W22Y和W22F取代，在Neuro-2a细胞中表达了GFP标记的构建体，并测量了它们对膜的易位性通过使用共聚焦显微镜对活细胞进行成像来对佛波酯进行处理。膜易位的程度依次为野生型> W22K> W22F> W22Y> W22A> W22D。野生型Munc13-1C1结构域及其突变体的佛波酯结合亲和力是磷脂酰丝氨酸（PS）依赖性的顺序，在20％和100％PS中野生型> W22K> W22A 100 W22D。对Munc13-1的佛波酯亲和力比C1域高。当Munc13-1易位到质膜时，C1结构域响应佛波酯而易位到内膜。分子动力学（80 ns）研究表明，Trp-22的柔韧性相对低于同源PKCδ和PKCθ的Trp-22。就Munc13-1C1域的整体负电荷状态及其与富含PS的质膜的可能相互作用讨论了结果。