School of Neurobiology, Biochemistry & Biophysics, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Ramat Aviv, Tel Aviv 69978, Israel.
Department of Oral Biology, The Goldschleger School of Dental Medicine, Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
Int J Mol Sci. 2024 Oct 16;25(20):11099. doi: 10.3390/ijms252011099.
Ubiquitin (Ub) signals are recognized and decoded into cellular responses by Ub-receptors, proteins that tether the Ub-binding domain(s) (UBDs) with response elements. Typically, UBDs bind mono-Ub in highly dynamic and weak affinity manners, presenting challenges in identifying and characterizing their binding interfaces. Here, we report the development of a new approach to facilitate the detection of these weak interactions using split-reporter systems where two interacting proteins are proximally co-translated from a single mRNA. This proximity significantly enhances the readout signals of weak protein-protein interactions (PPIs). We harnessed this system to characterize the ultra-weak UBD and ENTH (Epsin N-terminal Homology) and discovered that the yeast Ent1-ENTH domain contains two Ub-binding patches. One is similar to a previously characterized patch on STAM1(signal-transducing adaptor molecule)-VHS (Vps27, Hrs, and STAM), and the other was predicted by AlphaFold. Using a split-CAT selection system that co-translates Ub and ENTH in combination with mutagenesis, we assessed and confirmed the existence of a novel binding patch around residue F53 on ENTH. Co-translation in the split-CAT system provides an effective tool for studying weak PPIs and offers new insights into Ub-receptor interactions.
泛素 (Ub) 信号被 Ub 受体识别并解码为细胞反应，Ub 受体是将 Ub 结合域 (UBD) 与反应元件连接起来的蛋白质。通常，UBD 以高度动态和弱亲和力的方式结合单 Ub，这给鉴定和表征其结合界面带来了挑战。在这里，我们报告了一种新方法的发展，该方法使用分割报告系统来促进这些弱相互作用的检测，其中两个相互作用的蛋白质从单个 mRNA 近邻共翻译。这种接近显著增强了弱蛋白-蛋白相互作用 (PPI) 的读出信号。我们利用该系统对超弱 UBD 和 ENT（Epsin N-terminal Homology）进行了表征，并发现酵母 Ent1-ENTH 结构域包含两个 Ub 结合斑。一个类似于之前在 STAM1（信号转导衔接分子）-VHS（Vps27、Hrs 和 STAM）上表征的斑，另一个是由 AlphaFold 预测的。我们使用一种分割 CAT 选择系统，该系统将 Ub 和 ENTH 共翻译，并结合突变分析，评估并证实了 ENTH 上残基 F53 周围存在一个新的结合斑。在分割 CAT 系统中的共翻译为研究弱 PPI 提供了一种有效工具，并为 Ub 受体相互作用提供了新的见解。
