Time-Resolved Analysis of Protein-Protein Ensembles Using a Destabilizing Domain to Map Dynamic Interactions of...

Area of Science:

• Molecular Biology
• Virology
• Proteomics

Background:

• Dynamic protein-protein interactions are fundamental to cellular processes.
• Understanding the temporal order of these interactions is critical for deciphering biological mechanisms.
• Existing methods lack the temporal resolution to capture transient protein interactions.

Purpose of the Study:

• To develop a novel method for time-resolved analysis of protein-protein interactions.
• To investigate the temporal dynamics of protein interactions during viral infection.
• To identify novel protein interaction partners of SARS-CoV-2 nonstructural protein 15 (nsp15).

Main Methods:

• Development of the TRAPPED (time-resolved analysis of protein-protein ensembles) methodology.
• Utilizing a dihydrofolate reductase-destabilizing domain (DHFR(DD)) fused to proteins of interest.
• Employing trimethoprim (TMP) for temporal stabilization and Click chemistry for labeling and mass spectrometry.
• Evaluating protein interactions of SARS-CoV-2 nsp15 over a 2-hour period.

Main Results:

• Identified GEMIN5 and YBX3 as SARS-CoV-2 nsp15 interactors, linking them to viral RNA binding and infection regulation.
• Discovered CACYBP and FHL1 as nsp15 interactors, suggesting a role in disrupting host ERK1/2 signaling.
• Demonstrated that nsp15 interactions remain stable between 0 and 2 hours post-translation.

Conclusions:

• The TRAPPED methodology enables precise temporal mapping of protein-protein interactions.
• This approach provides new insights into the molecular mechanisms of SARS-CoV-2 infection.
• TRAPPED is applicable to studying other temporally regulated biological processes, including signal transduction.