Departments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
Proc Natl Acad Sci U S A. 2011 Aug 2;108(31):12723-8. doi: 10.1073/pnas.1105128108. Epub 2011 Jul 18.
SNARE proteins play a critical role in intracellular membrane fusion by forming tight complexes that bring two membranes together and involve sequences called SNARE motifs. These motifs have a high tendency to form amphipathic coiled-coils that assemble into four-helix bundles, and often precede transmembrane regions. NMR studies in dodecylphosphocholine (DPC) micelles suggested that the N-terminal half of the SNARE motif from the neuronal SNARE synaptobrevin binds to membranes, which appeared to contradict previous biophysical studies of synaptobrevin in liposomes. NMR analyses of synaptobrevin reconstituted into nanodiscs and into liposomes now show that most of its SNARE motif, except for the basic C terminus, is highly flexible, exhibiting cross-peak patterns and transverse relaxation rates that are very similar to those observed in solution. Considering the proximity to the bilayer imposed by membrane anchoring, our data show that most of the synaptobrevin SNARE motif has a remarkable reluctance to bind membranes. This conclusion is further supported by NMR experiments showing that the soluble synaptobrevin SNARE motif does not bind to liposomes, even though it does bind to DPC micelles. These results show that nanodiscs provide a much better membrane model than DPC micelles in this system, and that most of the SNARE motif of membrane-anchored synaptobrevin is accessible for SNARE complex formation. We propose that the charge and hydrophobicity of SNARE motifs is optimized to enable formation of highly stable SNARE complexes while at the same time avoiding membrane binding, which could hinder SNARE complex assembly.
SNARE 蛋白在细胞内膜融合中起着关键作用，它们形成紧密的复合物，将两个膜拉到一起，并涉及到称为 SNARE 基序的序列。这些基序具有形成两亲性卷曲螺旋的高倾向，这些螺旋组装成四螺旋束，并且通常先于跨膜区域。在十二烷基磷酸胆碱 (DPC) 胶束中的 NMR 研究表明，神经元 SNARE 突触融合蛋白的 SNARE 基序的 N 端半部分与膜结合，这似乎与先前在脂质体中对突触融合蛋白的生物物理研究相矛盾。现在，对重新构建到纳米盘和脂质体中的突触融合蛋白的 NMR 分析表明，除了碱性 C 端之外，其大部分 SNARE 基序都是高度灵活的，表现出与溶液中非常相似的交叉峰模式和横向弛豫率。考虑到膜锚定施加的双层接近度，我们的数据表明，突触融合蛋白的大部分 SNARE 基序非常不愿意与膜结合。这一结论进一步得到了 NMR 实验的支持，该实验表明可溶性突触融合蛋白 SNARE 基序即使与 DPC 胶束结合，也不会与脂质体结合。这些结果表明，在该系统中，纳米盘比 DPC 胶束提供了更好的膜模型，并且膜锚定的突触融合蛋白的大部分 SNARE 基序可用于 SNARE 复合物形成。我们提出，SNARE 基序的电荷和疏水性被优化，以能够形成高度稳定的 SNARE 复合物，同时避免与膜结合，这可能会阻碍 SNARE 复合物的组装。
