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

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Signaling

Background:

• Ubiquitin (Ub) signaling is crucial for cellular processes, mediated by Ub-receptors that bind Ub via Ubiquitin-Binding Domains (UBDs).
• Characterizing the weak and dynamic interactions between UBDs and Ub presents significant technical challenges.
• Understanding these interactions is key to deciphering Ub-receptor function in various cellular pathways.

Purpose of the Study:

• To develop and validate a novel split-reporter system for detecting and characterizing weak protein-protein interactions (PPIs).
• To apply this system to investigate the Ub-binding interfaces of the ENTH (Epsin N-terminal Homology) domain.
• To identify and characterize novel Ub-binding sites on the yeast Ent1-ENTH domain.

Main Methods:

• Development of a split-reporter system enabling proximal co-translation of interacting proteins from a single mRNA.
• Application of the split-CAT selection system for co-translating Ubiquitin and ENTH domain proteins.
• Utilizing mutagenesis combined with the split-CAT system to assess and confirm Ub-binding patches.

Main Results:

• The split-reporter system significantly enhances the signal readout for weak PPIs.
• The yeast Ent1-ENTH domain was found to possess two distinct Ub-binding patches.
• A novel Ub-binding patch around residue F53 on ENTH was identified and confirmed.
• One binding patch showed similarity to known sites on STAM1-VHS, while the second was predicted computationally.

Conclusions:

• Co-translation in split-reporter systems is an effective strategy for studying ultra-weak PPIs.
• This approach provides new insights into the molecular mechanisms of Ub-receptor interactions.
• The discovery of a novel binding site on ENTH expands our understanding of ubiquitin recognition.