Institut de Biologia Molecular de Barcelona, CSIC, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
BioNMR Laboratory, Inorganic and Organic Chemistry Department, Organic Chemistry Section, University of Barcelona, Parc Cientific de Barcelona, 08028 Barcelona, Spain.
Sci Rep. 2016 Jul 14;6:29232. doi: 10.1038/srep29232.
The activity of RING ubiquitin ligases (E3s) depends on an interaction between the RING domain and ubiquitin conjugating enzymes (E2), but posttranslational events or additional structural elements, yet largely undefined, are frequently required to enhance or regulate activity. Here, we show for the ubiquitin ligase RNF125 that, in addition to the RING domain, a C2HC Zn finger (ZnF) is crucial for activity, and a short linker sequence (Li2(120-128)) enhances activity. The contribution of these regions was first shown with truncated proteins, and the essential role of the ZnF was confirmed with mutations at the Zn chelating Cys residues. Using NMR, we established that the C2HC ZnF/Li2(120-128) region is crucial for binding of the RING domain to the E2 UbcH5a. The partial X-ray structure of RNF125 revealed the presence of extensive intramolecular interactions between the RING and C2HC ZnF. A mutation at one of the contact residues in the C2HC ZnF, a highly conserved M112, resulted in the loss of ubiquitin ligase activity. Thus, we identified the structural basis for an essential role of the C2HC ZnF and conclude that this domain stabilizes the RING domain, and is therefore required for binding of RNF125 to an E2.
RING 泛素连接酶 (E3s) 的活性取决于 RING 结构域与泛素连接酶 (E2) 的相互作用，但通常需要翻译后事件或其他结构元件来增强或调节活性。在这里，我们展示了泛素连接酶 RNF125，除了 RING 结构域外，C2HC Zn 指 (ZnF) 对于活性也是至关重要的，并且短的连接序列 (Li2(120-128)) 可以增强活性。这些区域的贡献首先通过截断蛋白显示出来，并且 Zn 螯合半胱氨酸残基的突变证实了 ZnF 的重要作用。使用 NMR，我们确定了 C2HC ZnF/Li2(120-128) 区域对于 RING 结构域与 E2 UbcH5a 的结合至关重要。RNF125 的部分 X 射线结构揭示了 RING 和 C2HC ZnF 之间存在广泛的分子内相互作用。C2HC ZnF 中的一个接触残基 (高度保守的 M112) 的突变导致泛素连接酶活性丧失。因此，我们确定了 C2HC ZnF 发挥重要作用的结构基础，并得出结论，该结构域稳定了 RING 结构域，因此是 RNF125 与 E2 结合所必需的。
