The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function.
The Nedd8 Non-covalent Binding Region in the Smurf HECT Domain is Critical to its Ubiquitn Ligase Function.
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DOI：
10.1038/srep41364
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发表时间：
2017-02-07
影响因子：
4.6
通讯作者：
Zhang L
中科院分区：
文献类型：
--
作者：
He S;Cao Y;Xie P;Dong G;Zhang L
NEDD8是一种泛素样蛋白，通过与靶蛋白的结合来控制重要的生物事件。我们以前发现了Hect-type泛素连接酶SMurf1，它控制着不同的细胞过程，是由Nedd8通过共价脱氢酶激活的。然而，非共价结合对Nedd8的影响仍不清楚。在这项研究中，我们证明了SMurf1和它的同系物SMurf2在其催化Hect结构域中都带有一个非共价Nedd8结合位点。结构分析表明，SMurf2在N叶的小亚区和Hect结构域的C叶具有Nedd8结合位点。有趣的是，一致的Nedd8结合序列L(X7)R(X5)F(X)ALQ在两个蓝精灵中都是保守的。突变研究表明，保守序列中的所有五个残基都是与Nedd8结合所必需的。功能研究表明，破坏蓝精灵与Nedd8相互作用的突变减少了Nedd8的碱基，稳定了蛋白质。此外，蓝精灵中的Nedd8结合位点是其泛素连接酶对底物的活性和自身泛素化所必需的。最后，我们发现Nedd8与S-MURF结合在调节细胞迁移以及骨形态发生蛋白和转化生长因子β信号通路中发挥重要作用。
Nedd8 is a ubiquitin-like protein that controls vital biological events through conjugation to target proteins. We previously identified the HECT-type ubiquitin ligase Smurf1 which controls diverse cellular processes is activated by Nedd8 through covalent neddylation. However, the effect of non-covalent binding to Nedd8 remains unknown. In this study, we demonstrate that both Smurf1 and its homologue Smurf2 carry a non-covalent Nedd8-binding site within its catalytic HECT domain. Structural analysis reveals that Smurf2 has Nedd8-binding sites within the small sub-domain of N-lobe and the C-lobe of HECT domain. Interestingly, the consensus Nedd8 binding sequence, L(X7)R(X5)F(X)ALQ is conserved in both Smurfs. Mutational studies reveal that all the five residues in the conserved sequence are required for binding to Nedd8. Functional studies suggest that mutations that disrupt Smurf interaction with Nedd8 reduce its neddylation and stabilize the protein. Furthermore, Nedd8 binding site in Smurf is shown to be necessary for its ubiquitin ligase activity towards the substrate and also the self-ubiquitylation. Finally, we show that Nedd8 binding to Smurf plays important roles in the regulation of cell migration and the BMP and TGFβ signaling pathways.