E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

中文翻译：

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非经典 E2 结合酶的发现和表征

E2 结合酶 (E2) 在导致泛素附着到底物的酶级联反应中发挥着核心作用。这个过程被称为泛素化，是维持细胞稳态所必需的，并影响几乎所有的细胞过程。通过与多个 E3 连接酶相互作用，E2 决定细胞内的泛素化景观。自发现以来，泛素化一直被认为是一种专门针对赖氨酸侧链的翻译后修饰（典型泛素化）。我们使用基质辅助激光解吸/电离飞行时间质谱法来鉴定和表征能够将泛素与丝氨酸和/或苏氨酸缀合的 E2 家族。我们使用结构建模和预测工具来确定这些 E2 用于与泛素及其底物相互作用的关键活性决定因素。我们的结果揭示了非规范泛素化所必需的缺失的 E2，强调了泛素修饰的适应性和多功能性。