SPINDLY (SPY) in Arabidopsis thaliana is a novel nucleocytoplasmic protein O-fucosyltransferase (POFUT), which regulates diverse developmental processes. Sequence analysis indicates that SPY is distinct from ER-localized POFUTs and contains N-terminal tetratricopeptide repeats (TPRs) and a C-terminal catalytic domain resembling the O-linked-N-acetylglucosamine (GlcNAc) transferases (OGTs). However, the structural feature that determines the distinct enzymatic selectivity of SPY remains unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of SPY and its complex with GDP-fucose, revealing distinct active-site features enabling GDP-fucose instead of UDP-GlcNAc binding. SPY forms an antiparallel dimer instead of the X-shaped dimer in human OGT, and its catalytic domain interconverts among multiple conformations. Analysis of mass spectrometry, co-IP, fucosylation activity, and cryo-EM data further demonstrates that the N-terminal disordered peptide in SPY contains trans auto-fucosylation sites and inhibits the POFUT activity, whereas TPRs 1–5 dynamically regulate SPY activity by interfering with protein substrate binding.

拟南芥中的 SPINDLY (SPY) 是一种新型核细胞质蛋白O-岩藻糖基转移酶 (POFUT)，可调节多种发育过程。序列分析表明，SPY 与 ER 定位的 POFUT 不同，并且包含 N 端四肽重复序列 (TPR) 和类似于O连接-N-乙酰氨基葡萄糖 (GlcNAc) 转移酶 (OGT) 的 C 端催化结构域。然而，决定 SPY 独特酶选择性的结构特征仍然未知。在这里，我们报道了 SPY 及其与 GDP-岩藻糖复合物的冷冻电子显微镜 (cryo-EM) 结构，揭示了独特的活性位点特征，使 GDP-岩藻糖而不是 UDP-GlcNAc 结合成为可能。 SPY形成反平行二聚体，而不是人OGT中的X形二聚体，并且其催化结构域在多种构象之间相互转换。质谱、co-IP、岩藻糖基化活性和冷冻电镜数据的分析进一步表明，SPY 中的 N 端无序肽含有反式自岩藻糖基化位点并抑制 POFUT 活性，而 TPRs 1-5 通过动态调节 SPY 活性干扰蛋白质底物结合。