Hubei Hongshan Laboratory, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China.
Nat Commun. 2022 Dec 2;13(1):7424. doi: 10.1038/s41467-022-35234-0.
Arabidopsis glycosyltransferase family 41 (GT41) protein SPINDLY (SPY) plays pleiotropic roles in plant development. Despite the amino acid sequence is similar to human O-GlcNAc transferase, Arabidopsis SPY has been identified as a novel nucleocytoplasmic protein O-fucosyltransferase. SPY-like proteins extensively exist in diverse organisms, indicating that O-fucosylation by SPY is a common way to regulate intracellular protein functions. However, the details of how SPY recognizes and glycosylates substrates are unknown. Here, we present a crystal structure of Arabidopsis SPY/GDP complex at 2.85 Å resolution. SPY adopts a head-to-tail dimer. Strikingly, the conformation of a 'catalytic SPY'/GDP/'substrate SPY' complex formed by two symmetry-related SPY dimers is captured in the crystal lattice. The structure together with mutagenesis and enzymatic data demonstrate SPY can fucosylate itself and SPY's self-fucosylation region negatively regulates its enzyme activity, reveal SPY's substrate recognition and enzyme mechanism, and provide insights into the glycan donor substrate selection in GT41 proteins.
拟南芥糖基转移酶家族 41（GT41）蛋白 SPINDLY（SPY）在植物发育中发挥着多种作用。尽管其氨基酸序列与人类 O-GlcNAc 转移酶相似，但拟南芥 SPY 已被鉴定为一种新型核质蛋白 O-岩藻糖基转移酶。SPY 样蛋白广泛存在于各种生物中，表明 SPY 的 O-岩藻糖基化是调节细胞内蛋白质功能的一种常见方式。然而，SPY 如何识别和糖基化底物的细节尚不清楚。在这里，我们展示了拟南芥 SPY/GDP 复合物的晶体结构，分辨率为 2.85 Å。SPY 采用头尾二聚体的形式。引人注目的是，由两个对称相关的 SPY 二聚体形成的“催化 SPY'/GDP/'底物 SPY”复合物的构象被捕捉在晶格中。该结构结合突变和酶学数据表明，SPY 可以自身岩藻糖基化，SPY 的自身岩藻糖基化区域负调控其酶活性，揭示了 SPY 的底物识别和酶机制，并为 GT41 蛋白中糖供体底物的选择提供了见解。
