Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Protein Sci. 2020 Jun;29(6):1440-1458. doi: 10.1002/pro.3844. Epub 2020 Mar 7.
Munc13-1 is crucial for neurotransmitter release and, together with Munc18-1, orchestrates assembly of the neuronal SNARE complex formed by syntaxin-1, SNAP-25, and synaptobrevin. Assembly starts with syntaxin-1 folded into a self-inhibited closed conformation that binds to Munc18-1. Munc13-1 is believed to catalyze the opening of syntaxin-1 to facilitate SNARE complex formation. However, different types of Munc13-1-syntaxin-1 interactions have been reported to underlie this activity, and the critical nature of Munc13-1 for release may arise because of its key role in bridging the vesicle and plasma membranes. To shed light into the mechanism of action of Munc13-1, we have used NMR spectroscopy, SNARE complex assembly experiments, and liposome fusion assays. We show that point mutations in a linker region of syntaxin-1 that forms intrinsic part of the closed conformation strongly impair stimulation of SNARE complex assembly and liposome fusion mediated by Munc13-1 fragments, even though binding of this linker region to Munc13-1 is barely detectable. Conversely, the syntaxin-1 SNARE motif clearly binds to Munc13-1, but a mutation that disrupts this interaction does not affect SNARE complex assembly or liposome fusion. We also show that Munc13-1 cannot be replaced by an artificial tethering factor to mediate liposome fusion. Overall, these results emphasize how very weak interactions can play fundamental roles in promoting conformational transitions and strongly support a model whereby the critical nature of Munc13-1 for neurotransmitter release arises not only from its ability to bridge two membranes but also from an active role in opening syntaxin-1 via interactions with the linker.
Munc13-1 对于神经递质释放至关重要，它与 Munc18-1 一起协调由突触融合蛋白 1（syntaxin-1）、突触相关蛋白 25（SNAP-25）和囊泡相关膜蛋白（synaptobrevin）组成的神经元 SNARE 复合物的组装。组装始于折叠成自我抑制的封闭构象的 syntaxin-1，该构象与 Munc18-1 结合。据信 Munc13-1 可催化 syntaxin-1 的打开，以促进 SNARE 复合物的形成。然而，已经报道了不同类型的 Munc13-1-syntaxin-1 相互作用是这种活性的基础，并且 Munc13-1 对释放的关键性质可能是由于其在桥接囊泡和质膜方面的关键作用。为了深入了解 Munc13-1 的作用机制，我们使用了 NMR 光谱学、SNARE 复合物组装实验和脂质体融合测定法。我们表明，在形成封闭构象的内在部分的 syntaxin-1 的连接区中的点突变强烈损害了由 Munc13-1 片段介导的 SNARE 复合物组装和脂质体融合的刺激，尽管该连接区与 Munc13-1 的结合几乎无法检测到。相反，syntaxin-1 SNARE 基序明显与 Munc13-1 结合，但破坏这种相互作用的突变不会影响 SNARE 复合物组装或脂质体融合。我们还表明，Munc13-1 不能被人工连接因子取代来介导脂质体融合。总体而言，这些结果强调了非常弱的相互作用如何在促进构象转变中发挥重要作用，并强烈支持这样一种模型，即 Munc13-1 对于神经递质释放的关键性质不仅来自其桥接两个膜的能力，还来自通过与连接子相互作用打开 syntaxin-1 的积极作用。
