Sequence determinants of E2-E6AP binding affinity and specificity
Sequence determinants of E2-E6AP binding affinity and specificity
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DOI：
10.1016/j.jmb.2007.03.026
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发表时间：
2007-06-01
影响因子：
5.6
通讯作者：
Kuhlman, Brian
中科院分区：
文献类型：
--
作者：
Eletr, Ziad M.;Kuhlman, Brian
泛素与底物的缀合需要由活化酶（EI）、缀合酶（E2）和连接酶（E3）组成的一系列酶促反应。用泛素标记适当的底物是通过特异性E2-E3和E3-底物相互作用实现的。E6 AP是E3的HECT家族的成员，此前已被证明与E2的UbcH 7和UbcH 8结合并发挥作用。为了破译这种特异性的序列决定簇，我们已经开发了一种基于荧光偏振的定量E2-E3结合测定，并使用该测定来测量野生型和突变体E2-E6 AP相互作用的亲和力。丙氨酸扫描的E6 AP-UbcH 7结合界面确定了UbcH 7上的四个侧链和E6 AP上的六个侧链，其对结合自由能的贡献超过1 kcal/mol。来自UbcH 7的两个热点残基（K96和K100）在UbcH 8中是保守的，但在其他E2中有所不同。为了确定这些是否是关键的特异性决定残基，我们试图通过将UbcH 5 b中的相应位置突变为赖氨酸残基来诱导E2 UbcH 5 b和E6 AP之间的更紧密的关联。令人惊讶的是，突变几乎没有影响，而是在UbcH 7位置4处的突变，其不在UbcH 7-E6 AP界面上的热点处，显著增强了UbcH 5 bs对E6 AP的亲和力。该结果表明，E2-E3结合特异性是促进结合的有利相互作用和防止与不需要的配偶体结合的不利相互作用的函数。(C)2007爱思唯尔有限公司保留所有权利。
The conjugation of ubiquitin to substrates requires a series of enzymatic reactions consisting of an activating enzyme (EI), conjugating enzymes (E2) and ligases (E3). Tagging the appropriate substrate with ubiquitin is achieved by specific E2-E3 and E3-substrate interactions. E6AP, a member of the HECT family of E3s, has been previously shown to bind and function with the E2s UbcH7 and UbcH8. To decipher the sequence determinants of this specificity we have developed a quantitative E2-E3 binding assay based on fluorescence polarization and used this assay to measure the affinity of wild-type and mutant E2-E6AP interactions. Alanine scanning of the E6AP-UbcH7 binding interface identified four side-chains on UbcH7 and six side-chains on E6AP that contribute more than 1 kcal/mol to the binding free energy. Two of the hot spot residues from UbcH7 (K96 and K100) are conserved in UbcH8 but vary across other E2s. To determine if these are key specificity determining residues, we attempted to induce a tighter association between the E2 UbcH5b and E6AP by mutating the corresponding positions in UbcH5b to lysine residues. Surprisingly, the mutations had little effect, but rather a mutation at UbcH7 position 4, which is not at a hot spot on the UbcH7-E6AP interface, significantly strengthened UbcH5bs affinity for E6AP. This result indicates that E2-E3 binding specificities are a function of both favorable interactions that promote binding, and unfavorable interactions that prevent binding with unwanted partners. (C) 2007 Elsevier Ltd. All rights reserved.