Institute of Molecular Cell and Systems Biology, College of Medical Veterinary and Life Sciences, University of Glasgow, Glasgow, UK.
MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, UK.
Nat Struct Mol Biol. 2021 Apr;28(4):356-364. doi: 10.1038/s41594-021-00576-8. Epub 2021 Apr 1.
Ubiquitin-specific protease 1 (USP1) acts together with the cofactor UAF1 during DNA repair processes to specifically remove monoubiquitin signals. One substrate of the USP1-UAF1 complex is the monoubiquitinated FANCI-FANCD2 heterodimer, which is involved in the repair of DNA interstrand crosslinks via the Fanconi anemia pathway. Here we determine structures of human USP1-UAF1 with and without ubiquitin and bound to monoubiquitinated FANCI-FANCD2. The crystal structures of USP1-UAF1 reveal plasticity in USP1 and key differences to USP12-UAF1 and USP46-UAF1, two related proteases. A cryo-EM reconstruction of USP1-UAF1 in complex with monoubiquitinated FANCI-FANCD2 highlights a highly orchestrated deubiquitination process, with USP1-UAF1 driving conformational changes in the substrate. An extensive interface between UAF1 and FANCI, confirmed by mutagenesis and biochemical assays, provides a molecular explanation for the requirement of both proteins, despite neither being directly involved in catalysis. Overall, our data provide molecular details of USP1-UAF1 regulation and substrate recognition.
泛素特异性蛋白酶 1（USP1）在 DNA 修复过程中与共因子 UAF1 一起作用，特异性去除单泛素信号。USP1-UAF1 复合物的一个底物是单泛素化的 FANCI-FANCD2 异二聚体，它通过范可尼贫血途径参与 DNA 链间交联的修复。在这里，我们确定了人 USP1-UAF1 与泛素结合和与单泛素化的 FANCI-FANCD2 结合的结构。USP1-UAF1 的晶体结构揭示了 USP1 的可塑性，以及与 USP12-UAF1 和 USP46-UAF1 的关键差异，这两种相关的蛋白酶。与单泛素化的 FANCI-FANCD2 结合的 USP1-UAF1 的冷冻电镜重建突出了高度协调的去泛素化过程，USP1-UAF1 驱动底物的构象变化。UAF1 和 FANCI 之间广泛的界面，通过突变和生化分析得到证实，为这两种蛋白的需求提供了分子解释，尽管它们都不直接参与催化。总的来说，我们的数据提供了 USP1-UAF1 调节和底物识别的分子细节。
