The Ubiquitin Binding Region of the Smurf HECT Domain Facilitates Polyubiquitylation and Binding of Ubiquitylated Substrates
The Ubiquitin Binding Region of the Smurf HECT Domain Facilitates Polyubiquitylation and Binding of Ubiquitylated Substrates
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DOI：
10.1074/jbc.m109.044537
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发表时间：
2010-02-26
影响因子：
4.8
通讯作者：
Wrana, Jeffrey L.
中科院分区：
文献类型：
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作者：
Ogunjimi, Abiodun A.;Wiesner, Silke;Wrana, Jeffrey L.
蛋白质的单泛素化和多泛素化是广泛的生物过程中的关键步骤。然而，介导这些不同事件的分子机制却知之甚少。在这里，我们利用核磁共振波谱在Smurf泛素连接酶HECT结构域上绘制了一个非共价泛素结合表面（UBS）。对HECT UBS突变体的分析表明，干扰UBS表面可阻断细胞中smurf依赖性底物RhoA的降解。体外分析表明，UBS不需要ubch7依赖于HECT催化半胱氨酸的充电。令人惊讶的是，尽管UBS对于Smurf本身和Smurf底物RhoA的多泛素化都是必需的，但对于单泛素化却不是必需的。此外，我们发现UBS的突变干扰了RhoA单双向化形式与Smurf HECT结构域的有效结合。我们的研究结果表明，UBS通过稳定泛素化底物与HECT结构域的结合来促进多泛素化。
Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monou-biquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.