Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases
Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases
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DOI：
10.15252/embj.201694314
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发表时间：
2017-02-15
期刊：
影响因子：
11.4
通讯作者：
Prag, Gali
中科院分区：
文献类型：
--
作者：
Attali, Ilan;Tobelaim, William Sam;Prag, Gali
泛素化控制蛋白质的功能和降解。因此，需要严格控制泛素连接酶。我们发现了Nedd 4连接酶的进化保守的变构抑制机制，并证明了其对不同底物的功能：酵母可溶性蛋白Rpn 10和Rvs 167，以及人受体酪氨酸激酶FGFR 1和心脏I-KS钾通道。我们发现，一个潜在的三聚化接口在结构上被HECT结构域α 1-螺旋阻断，其进一步在保守的赖氨酸残基上进行泛素化。遗传学、生物信息学、生物化学和生物物理学数据表明，这种α 1-缀合的泛素和HECT泛素结合补丁之间的吸引力将α 1-螺旋拉出界面，从而促进三聚化。引人注目的是，三聚化使连接酶失活。泛素化赖氨酸的精氨酸取代削弱了这种失活机制，并导致细胞中不受限制的FGFR 1泛素化。类似地，电生理数据和TIRF显微镜显示NEDD 4无限制突变体组成性下调I-KS通道，从而证实E3连接酶自抑制的功能重要性。
Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac I-KS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain alpha 1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this alpha 1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the alpha 1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the I-KS channel, thus confirming the functional importance of E3-ligase autoinhibition.