A site-directed mutagenesis study of the MdmX RING domain
A site-directed mutagenesis study of the MdmX RING domain
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DOI：
10.1016/j.bbrc.2014.04.065
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发表时间：
2014-05-16
影响因子：
3.1
通讯作者：
Sheng, Yi
中科院分区：
文献类型：
--
作者：
Egorova, Olga;Sheng, Yi
Mdm2 和 MdmX 是肿瘤抑制因子 p53 的重要负调节因子。结构同源的 Mdm2 和 MdmX 通过直接阻断 p53 转录激活来抑制 p53。 Mdm2 还通过其 C 端 RING 结构域介导的 E3 连接酶活性来修饰和靶向 p53，以实现 265 蛋白酶体依赖性蛋白质降解。然而，MdmX 缺乏内在的 E3 连接酶活性，尽管含有保守的 RING 结构域，但无法催化 p53 泛素化。因此，Mdm2 和 MdmX RING 结构域之间的比较结构分析提供了一种独特的方法来阐明两种蛋白质在泛素化中的不同功能。我们对 MdmX RING 结构域进行了定点诱变，发现残基 N448 替换为半胱氨酸，残基 K478 替换为精氨酸，赋予了 MdmX RING 结构域泛素化活性。 Mdm2和MdmX RING结构域的结构分析表明，位于Mdm2 RING二聚体界面的Mdm2残基C449（与MdmX RING N448结构同源）对于RING二聚体结构的稳定性至关重要，而残基R479（与MdmX RING K478结构同源）在招募和激活泛素E2中发挥作用结合酶。这项研究为 Mdm2 RING 结构域介导的泛素化分子机制提供了新的见解。 (C) 2014 Elsevier Inc. 保留所有权利。
Mdm2 and MdmX are important negative regulators of the tumor suppressor p53. Structurally homologous Mdm2 and MdmX inhibit p53 by directly blocking p53 transcriptional activation. Mdm2 also modifies and targets p53 for 265 proteasome dependent protein degradation through E3 ligase activity mediated by its C-terminal RING domain. However, MdmX lacks intrinsic E3 ligase activity and fails to catalyze ubiquitination of p53 despite containing a conserved RING domain. Thus, a comparative structural analysis between the Mdm2 and MdmX RING domains offers a unique way to elucidate the distinct functions of the two proteins in ubiquitination. We performed site-directed mutagenesis of the MdmX RING domain and found that the substitution of the residue N448 for cysteine and the substitution of the residue K478 for arginine granted MdmX RING domain ubiquitination activity. The structural analysis of the Mdm2 and MdmX RING domains revealed that the residue C449 of Mdm2 (structurally homologous to MdmX RING N448) located at the Mdm2 RING dimer interface is critical for the stability of the RING dimer structure, while the residue R479 (structurally homologous to MdmX RING K478) plays a role in recruiting and activating the ubiquitin E2 conjugating enzyme. This study provides new insight into the molecular mechanism of Mdm2 RING domain mediated ubiquitination. (C) 2014 Elsevier Inc. All rights reserved.