@article {Kordonsky2024-dp,
	title = {Proximal co-translation facilitates detection of weak protein-protein interactions},
	journal = {Int. J. Mol. Sci.},
	volume = {25},
	number = {20},
	year = {2024},
	month = {oct},
	abstract = {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.},
	keywords = {bacterial selection, post-translation modification, protein-protein interaction, split chloramphenicol acetyltransferase, ubiquitin receptor},
	author = {Kordonsky, Alina and Gabay, Matan and Rosinoff, Aurelia and Avishid, Reut and Flornetin, Amir and Deouell, Noam and Abd Alkhaleq, Taimaa and Efron, Noa and Milshtein, Shoham and Shifman, Julia M and Gal, Maayan and Prag, Gali}
}
