ETH Zurich, Institute of Molecular Biology & Biophysics, Zurich, Switzerland.
University of California, San Francisco, USA.
Nat Commun. 2023 Aug 29;14(1):5266. doi: 10.1038/s41467-023-40807-8.
Pupylation, a post-translational modification found in Mycobacterium tuberculosis and other Actinobacteria, involves the covalent attachment of prokaryotic ubiquitin-like protein (Pup) to lysines on target proteins by the ligase PafA (proteasome accessory factor A). Pupylated proteins, like ubiquitinated proteins in eukaryotes, are recruited for proteasomal degradation. Proteomic studies suggest that hundreds of potential pupylation targets are modified by the sole existing ligase PafA. This raises intriguing questions regarding the selectivity of this enzyme towards a diverse range of substrates. Here, we show that the availability of surface lysines alone is not sufficient for interaction between PafA and target proteins. By identifying the interacting residues at the pupylation site, we demonstrate that PafA recognizes authentic substrates via a structural recognition motif centered around exposed lysines. Through a combination of computational analysis, examination of available structures and pupylated proteomes, and biochemical experiments, we elucidate the mechanism by which PafA achieves recognition of a wide array of substrates while retaining selective protein turnover.
泛素化，一种在结核分枝杆菌和其他放线菌中发现的翻译后修饰过程，涉及通过连接酶 PafA（蛋白酶体辅助因子 A）将原核泛素样蛋白（Pup）共价连接到靶蛋白上的赖氨酸。与真核生物中的泛素化蛋白一样，泛素化蛋白被招募进行蛋白酶体降解。蛋白质组学研究表明，仅存在的连接酶 PafA 就可以修饰数百种潜在的泛素化靶标。这就提出了一个有趣的问题，即该酶对各种底物的选择性。在这里，我们表明表面赖氨酸的可用性本身不足以进行 PafA 与靶蛋白之间的相互作用。通过鉴定泛素化位点的相互作用残基，我们证明 PafA 通过以暴露的赖氨酸为中心的结构识别基序识别真实的底物。通过计算分析、可用结构和泛素化蛋白质组的检查以及生化实验的结合，我们阐明了 PafA 如何在保留选择性蛋白质周转的同时实现对广泛底物的识别的机制。
