Dynamic conformational changes in munc18 prevent syntaxin binding.
Dynamic conformational changes in munc18 prevent syntaxin binding.
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DOI：
10.1371/journal.pcbi.1001097
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发表时间：
2011-03
影响因子：
4.3
通讯作者：
Ashery U
中科院分区：
文献类型：
--
作者：
Bar-On D;Nachliel E;Gutman M;Ashery U
Sec1/munc18蛋白家族通过与SNARE蛋白的结合在真核细胞中实现囊泡融合是必不可少的。蛋白激酶C通过磷酸化munc18a来调节这些相互作用，从而降低其对中心SNARE成员之一syntaxin-1a的亲和力。已建立的假设是，磷酸化的munc18a对syntaxin-1a的亲和力降低是两种蛋白质之间局部静电排斥的结果，这干扰了它们的相容性。目前的研究挑战了这一范式，并通过揭示由拟磷突变诱导的munc18a的syntaxin-non-binding构象提供了一种新的机制解释。在本研究中，我们利用分子动力学模拟，探讨了野生型munc18a与拟磷突变体munc18a的动力学关系。我们关注的是在结构域3a和1之间的空腔中发生的结构变化，这是主要的句法结合位点。模拟结果表明，自由野生型munc18a在其空腔（主要语法蛋白结合位点）大小不同的几个构象之间表现出动态平衡。腔体大小的灵活性可能有助于句法蛋白的结合或解结合。在硅中，将拟磷突变插入到munc18a结构中诱导形成一种构象，其中syntaxin结合区域是刚性的，并且由于位于腔两侧的残基之间的相互作用而被阻断。因此，我们认为，拟磷突变体/磷酸化munc18a的亲和力降低是封闭腔构象的结果，这使得syntaxin在能量和空间上都不适合结合。目前的研究表明，磷酸化是一个重要的生物学过程，可以作为蛋白质显著构象变化的驱动力，调节它们对靶蛋白的亲和力。蛋白质磷酸化通过激活或失活蛋白质，在参与信号转导或细胞过程协调的多组分系统中起着重要的调节作用。磷酸化诱导蛋白质实质性构象变化的潜力，从而改变其对靶蛋白的亲和力，已经被证明，但这一过程的动力学尚未完全阐明。在本研究中，我们通过分子动力学模拟研究了munc18a的动态构象变化，munc18a是一种对神经递质释放至关重要并与SNARE syntaxin-1紧密相互作用的蛋白质。我们进一步研究了munc18a在磷酸化时发生的构象变化，降低了它对syntaxin-1a的亲和力。模拟结果表明，syntaxin-unbounded munc18a具有构象灵活性，可以改变syntaxin-1a结合腔的形状。在硅中，将拟磷突变插入到munc18a中导致合成素结合位点的灵活性和关闭性降低。我们认为磷酸化的munc18a与syntaxin-1a的亲和力降低源于syntaxin-1a难以与munc18a闭合腔构象结合，这是由munc18a的PKC磷酸化引起的。
The Sec1/munc18 protein family is essential for vesicle fusion in eukaryotic cells via binding to SNARE proteins. Protein kinase C modulates these interactions by phosphorylating munc18a thereby reducing its affinity to one of the central SNARE members, syntaxin-1a. The established hypothesis is that the reduced affinity of the phosphorylated munc18a to syntaxin-1a is a result of local electrostatic repulsion between the two proteins, which interferes with their compatibility. The current study challenges this paradigm and offers a novel mechanistic explanation by revealing a syntaxin-non-binding conformation of munc18a that is induced by the phosphomimetic mutations. In the present study, using molecular dynamics simulations, we explored the dynamics of the wild-type munc18a versus phosphomimetic mutant munc18a. We focused on the structural changes that occur in the cavity between domains 3a and 1, which serves as the main syntaxin-binding site. The results of the simulations suggest that the free wild-type munc18a exhibits a dynamic equilibrium between several conformations differing in the size of its cavity (the main syntaxin-binding site). The flexibility of the cavity's size might facilitate the binding or unbinding of syntaxin. In silico insertion of phosphomimetic mutations into the munc18a structure induces the formation of a conformation where the syntaxin-binding area is rigid and blocked as a result of interactions between residues located on both sides of the cavity. Therefore, we suggest that the reduced affinity of the phosphomimetic mutant/phosphorylated munc18a is a result of the closed-cavity conformation, which makes syntaxin binding energetically and sterically unfavorable. The current study demonstrates the potential of phosphoryalation, an essential biological process, to serve as a driving force for dramatic conformational changes of proteins modulating their affinity to target proteins. Protein phosphorylation plays a significant regulatory role in multi-component systems engaged in signal transduction or coordination of cellular processes, by activating or deactivating proteins. The potential of phosphorylation to induce substantial conformational changes in proteins, thereby changing their affinity to target proteins, has already been shown but the dynamics of the process is not fully elucidated. In the present study, we investigated, by molecular dynamics simulations, the dynamic conformational changes in munc18a, a protein that is crucial for neurotransmitter release and interacts tightly with the SNARE syntaxin-1. We further investigated the conformational changes that occur in munc18a when it is phosphorylated, reducing its affinity to syntaxin-1a. The results of the simulations suggest that there is a conformational flexibility of the syntaxin-unbounded munc18a that allows changes in the shape of the syntaxin-1a binding cavity. In silico insertion of phosphomimetic mutations into munc18a led to a reduction in the flexibility and closure of the syntaxin-binding site. We suggest that the reduced affinity of phosphorylated munc18a to syntaxin-1a stems from the difficulty of syntaxin-1a to bind to the munc18a closed-cavity conformation, induced by the PKC phosphorylation of munc18a.
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