Forschungszentrum Jülich, Structural Biochemistry (IBI-7) and Jülich Center for Structural Biology (JuStruct), Wilhelm-Johnen-Straße, 52425 Jülich, Germany; Heinrich Heine University of Düsseldorf, Physical Biology, Universitätsstraße 1, 40225 Düsseldorf, Germany.
Max-Planck Institute for Multidisciplinary Sciences, Dept. of Neurobiology, Am Faßberg 11, 37077 Göttingen, Germany.
J Mol Biol. 2023 May 15;435(10):168069. doi: 10.1016/j.jmb.2023.168069. Epub 2023 Mar 30.
The neuronal SNARE protein SNAP25a (isoform 2) forms part of the SNARE complex eliciting synaptic vesicle fusion during neuronal exocytosis. While the post-fusion cis-SNARE complex has been studied extensively, little is known about the pre-fusion conformation of SNAP25a. Here we analyze monomeric SNAP25a by NMR spectroscopy, further supported by small-angle X-ray scattering (SAXS) experiments. SAXS data indicate that monomeric SNAP25 is more compact than a Gaussian chain but still a random coil. NMR shows that for monomeric SNAP25a, before SNAP25a interacts with its SNARE partners to drive membrane fusion, only the N-terminal part (region A5 to V36) of the first SNARE motif, SN1 (L11 - L81), is helical, comprising two α-helices (ranging from A5 to Q20 and S25 toV36). From E37 onwards, SNAP25a is mostly disordered and displays high internal flexibility, including the C-terminal part of SN1, almost the entire second SNARE motif (SN2, N144-A199), and the connecting loop region. Apart from the N-terminal helices, only the C-termini of both SN1 (E73 - K79) and SN2 (region T190 - A199), as well as two short regions in the connecting loop (D99 - K102 and E123 - M127) show a weak α-helical propensity (α-helical population < 25%). We speculate that the N-terminal helices (A5 to Q20 and S25 to V36) which constitute the N-terminus of SN1 act as a nucleation site for initiating SNARE zippering.
神经元 SNARE 蛋白 SNAP25a（同种型 2）是 SNARE 复合物的一部分，在神经元胞吐过程中引发突触小泡融合。虽然融合后的顺式 SNARE 复合物已经得到了广泛的研究，但对于 SNAP25a 的预融合构象知之甚少。在这里，我们通过 NMR 光谱学分析单体 SNAP25a，进一步得到小角度 X 射线散射（SAXS）实验的支持。SAXS 数据表明，单体 SNAP25 比高斯链更紧凑，但仍然是无规卷曲。NMR 表明，对于单体 SNAP25a，在 SNAP25a 与其 SNARE 伙伴相互作用以驱动膜融合之前，只有第一个 SNARE 模体（SN1，L11-L81）的 N 端部分（A5 到 V36 区域）是螺旋的，由两个α-螺旋组成（范围从 A5 到 Q20 和 S25 到 V36）。从 E37 开始，SNAP25a 主要是无序的，表现出高度的内部灵活性，包括 SN1 的 C 端部分，几乎整个第二个 SNARE 模体（SN2，N144-A199），以及连接环区。除了 N 端螺旋外，只有 SN1 的 C 端（E73-K79）和 SN2 的 C 端（区域 T190-A199），以及连接环中的两个短区域（D99-K102 和 E123-M127）显示出较弱的α-螺旋倾向（α-螺旋含量<25%）。我们推测，构成 SN1 N 端的 N 端螺旋（A5 到 Q20 和 S25 到 V36）充当 SNARE 拉链的起始核位点。
