Proximal Co-Translation Facilitates Detection of Weak Protein-Protein Interactions

Alina Kordonsky, Matan Gabay, Aurelia Rosinoff, Reut Avishid, Amir Flornetin, Noam Deouell, Taimaa Abd Alkhaleq, Noa Efron, Shoham Milshtein, Julia M. Shifman, Maayan Gal, Gali Prag*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish
Article number11099
JournalInternational Journal of Molecular Sciences
Volume25
Issue number20
DOIs
StatePublished - Oct 2024

Bibliographical note

Publisher Copyright:
© 2024 by the authors.

Keywords

  • bacterial selection
  • post-translation modification
  • protein–protein interaction
  • split chloramphenicol acetyltransferase
  • ubiquitin receptor

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