Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.
Proc Natl Acad Sci U S A. 2013 Apr 2;110(14):E1263-72. doi: 10.1073/pnas.1303309110. Epub 2013 Mar 18.
Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.
尽管泛素化在几乎所有细胞过程中都起着关键作用，但泛素（Ub）转移的机制细节仍在确定中。为了确定 E3 连接酶中调节活性的分子决定因素，我们对近 100,000 种蛋白质变体的库中的每个成员进行了评分，这些变体是在 E2 UbcH5c 的存在下，对鼠类泛素化因子 E4B（Ube4b）U 盒域的自身泛素化活性进行评分。该测定法鉴定了在体外和细胞 p53 降解测定中均增强活性的突变。具有活性增强作用的突变分为两种不同的机制类别：一种增加 U 盒：E2 结合亲和力，另一种变构刺激 E2∼Ub 缀合物的催化活性构象的形成。相同的突变在另一种 E2（Ube2w）存在下增强了 E3 的活性，这意味着存在共同的变构机制，因此我们的观察结果对其他 E3 具有普遍适用性。将 E3 活性与两种不同的 E2 进行比较，确定了另一个表现出 E3：E2 特异性的变体。我们的结果强调了高通量诱变在阐明酶活性的分子基础方面的普遍适用性。
