Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
PLoS Comput Biol. 2011 May;7(5):e1002056. doi: 10.1371/journal.pcbi.1002056. Epub 2011 May 26.
E2 ubiquitin-conjugating enzymes are crucial mediators of protein ubiquitination, which strongly influence the ultimate fate of the target substrates. Recently, it has been shown that the activity of several enzymes of the ubiquitination pathway is finely tuned by phosphorylation, an ubiquitous mechanism for cellular regulation, which modulates protein conformation. In this contribution, we provide the first rationale, at the molecular level, of the regulatory mechanism mediated by casein kinase 2 (CK2) phosphorylation of E2 Cdc34-like enzymes. In particular, we identify two co-evolving signature elements in one of the larger families of E2 enzymes: an acidic insertion in β4α2 loop in the proximity of the catalytic cysteine and two conserved key serine residues within the catalytic domain, which are phosphorylated by CK2. Our investigations, using yeast Cdc34 as a model, through 2.5 µs molecular dynamics simulations and biochemical assays, define these two elements as an important phosphorylation-controlled switch that modulates opening and closing of the catalytic cleft. The mechanism relies on electrostatic repulsions between a conserved serine phosphorylated by CK2 and the acidic residues of the β4α2 loop, promoting E2 ubiquitin charging activity. Our investigation identifies a new and unexpected pivotal role for the acidic loop, providing the first evidence that this loop is crucial not only for downstream events related to ubiquitin chain assembly, but is also mandatory for the modulation of an upstream crucial step of the ubiquitin pathway: the ubiquitin charging in the E2 catalytic cleft.
E2 泛素连接酶是蛋白质泛素化的关键介质，强烈影响靶底物的最终命运。最近，已经表明，泛素化途径的几种酶的活性通过磷酸化精细调节，磷酸化是一种普遍的细胞调节机制，调节蛋白质构象。在本研究中，我们在分子水平上首次提供了由酪蛋白激酶 2（CK2）磷酸化 E2 Cdc34 样酶介导的调节机制的基本原理。特别是，我们在 E2 酶的较大家族之一中鉴定了两个共同进化的特征元素：在靠近催化半胱氨酸的β4α2 环中的酸性插入和催化结构域内的两个保守关键丝氨酸残基，这些残基被 CK2 磷酸化。我们使用酵母 Cdc34 作为模型，通过 2.5µs 分子动力学模拟和生化测定进行的研究，将这两个元素定义为一个重要的磷酸化控制开关，调节催化裂缝的开闭。该机制依赖于 CK2 磷酸化的保守丝氨酸与β4α2 环的酸性残基之间的静电排斥，从而促进 E2 泛素充电活性。我们的研究确定了酸性环的一个新的和意外的关键作用，提供了第一个证据表明，该环不仅对于与泛素链组装相关的下游事件至关重要，而且对于泛素途径的一个上游关键步骤的调节也是强制性的：在 E2 催化裂缝中泛素的充电。
