Data from: Functional diversity and structural disorder in the human ubiquitination pathway

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred – E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions.泛素-蛋白酶体系统（ubiquitin-proteasome system）在细胞调控与蛋白质质量控制（protein quality control, PQC）中发挥核心作用。该系统以复杂度逐层提升的金字塔结构构建，包含2种E1（泛素激活酶，ubiquitin activating）、数十种E2（泛素结合酶，ubiquitin conjugating）以及数百种E3（泛素连接酶，ubiquitin ligase）。我们从KEGG BRITE数据库及相关文献中收集并分析E3序列，构建了包含563个人类E3的一致性数据集，并对其多项物理特征展开系统性分析。借助多种无序性预测工具（如IUPred）分析发现，该系统的结构无序程度呈上升趋势：E1为5.97%，E2为17.74%，E3为20.03%。能够同时结合E2与底物的E3（单亚基E3，single subunit E3, ssE3），其无序程度（22.98%）显著高于功能分区的E3：这类E3中，E2结合结构域（多环指结构域，multi RING-finger, mRF，0.62%）、支架结构域（6.01%）以及底物结合结构域（接头/底物识别亚基，adaptor/substrate recognition subunits，17.33%）彼此分离。在单亚基E3中，无序区域主要定位于底物/接头结合结构域，而结合E2的RING/HECT结构域则呈有序构象。为验证无序性在E3功能中的作用，我们通过简正模式分析与分子动力学模拟，揭示了人类CBL蛋白（一种参与调控多种酪氨酸激酶介导信号通路的E3）中一段高度柔性的无序连接肽，如何通过长距离构象变化使底物结合域与E2结合域相互靠近，进而辅助泛素转移过程。根据STRING数据库数据，拥有多个相互作用伴侣的E3（枢纽蛋白hub，22.90%）同样具有更高的无序性，显著高于非枢纽蛋白（non-hubs，18.36%）。此外，在蛋白质数据库（Protein Data Bank, PDB）的检索结果中，共发现21种不同的人类E3相互作用复合体，其中7种复合体里E3的无序区域会在结合伴侣后发生诱导折叠（或相互诱导折叠）。综上，本研究数据表明，结构无序性在E3连接酶的功能中扮演核心角色，具体体现于底物/接头结合功能，以及通过长距离构象转变实现泛素转移的机制中。