Structure and function of ubiquitin conjugating enzyme E2-25K: The tail is a core-dependent activity element
Structure and function of ubiquitin conjugating enzyme E2-25K: The tail is a core-dependent activity element
复制标题
DOI：
10.1021/bi970750u
复制
发表时间：
1997-08-26
期刊：
影响因子：
2.9
通讯作者：
Pickart, CM
中科院分区：
文献类型：
--
作者：
Haldeman, MT;Xia, G;Pickart, CM
泛素结合酶（E2）保守家族的单个成员介导泛素化和泛素依赖性降解途径中特定底物的周转。E2蛋白有一个高度保守的核心结构域，近似150个氨基酸，其中包含活性位点Cys;某些E2具有独特的末端延伸，被认为通过与底物或与反式作用因子如泛素-蛋白连接酶（E3）相互作用来促进选择性E2功能。我们使用哺乳动物泛素结合酶E2- 25 K在生化测试这一假设。两个截短衍生物的性质表明，E2- 25 K的47个残基的尾巴是必要的三个酶的特性：高活性的非锚定K48连接的聚泛素链的合成;活性位点Cys残基烷基化的阻力;和一个不寻常的歧视对noncognate（非哺乳动物）泛素激活（El）酶。然而，尾部不足以产生这些性质，如嵌合酶的特征所示，其中E3- 25 K的尾部融合到酵母UBC 4的核心结构域。这些和其他结果表明，尾部的特异性生化功能强烈依赖于E2- 25 k核心结构域的独特特征。因此，E2蛋白的保守核心结构域内的不同区域可能对功能非常重要。截短的E2- 25 K作为谷胱甘肽S-转移酶（GST）融合蛋白的表达导致E2- 25 K特异性性质的明显恢复，包括链合成活性。然而，截短的融合蛋白所利用的催化机制被证明是不同于野生型酶所利用的机制。融合蛋白的意外性质是由于GST诱导的二聚化。这些结果表明，潜在的自我协会，以调节多聚泛素链的合成活动的E2蛋白，并表明，应谨慎适用于解释GST融合蛋白的活动。
Individual members of the conserved family of ubiquitin conjugating enzymes (E2s) mediate the ubiquitination and turnover of specific substrates of the ubiquitin-dependent degradation pathway. E2 proteins have a highly conserved core domain of similar to 150 amino acids which contains the active-site Cys; Certain E2s have unique terminal extensions, which are thought to contribute to selective E2 function by interacting either with substrates or with trans-acting factors such as ubiquitin-protein ligases (E3s). We used the mammalian ubiquitin conjugating enzyme E2-25K in a biochemical test of this hypothesis. The properties of two truncated derivatives show that the 47-residue tail of E2-25K is necessary for three of the enzyme's characteristic properties: high activity in the synthesis of unanchored K48-linked polyubiquitin chains; resistance of the active-site Cys residue to alkylation; and an unusual discrimination against noncognate (nonmammalian) ubiquitin activating (El) enzymes. However, the tail is not sufficient to generate these properties, as shown by the characteristics of a chimeric enzyme in which the tail of E3-25K was fused to the core domain of yeast UBC4. These and other results indicate that the specific biochemical function of the tail is strongly dependent upon unique features of the E2-25k core domain. Thus, divergent regions within the conserved core domains of E2 proteins may be highly significant for function. Expression of truncated E2-25K as a glutathione S-transferase (GST) fusion protein resulted in the apparent recovery of E2-25K-specific properties, including activity in chain synthesis. However, the catalytic mechanism utilized by the truncated fusion protein proved to be distinct from the mechanism utilized by the wild-type enzyme. The unexpected properties of the fusion protein were due to GST-induced dimerization. These results indicate the potential for self-association to modulate the polyubiquitin chain synthesis activities of E2 proteins, and indicate that caution should be applied in interpreting the activities of GST fusion proteins.