Time-Resolved Analysis of Protein–Protein Ensembles Using a Destabilizing Domain to Map Dynamic Interactions of SARS-CoV-2 nsp15

Dynamic protein-protein interactions are key drivers of many cellular processes. Determining the relative sequence and precise timing of these interactions is crucial for elucidating the functional dynamics of biological processes. Here, we developed a time-resolved analysis of protein-protein ensembles using a destabilizing domain (TRAPPED) to study protein-protein interactions in a temporal manner. We have taken advantage of a dihydrofolate reductase-destabilizing domain (DHFR(DD)) that can be fused to a protein of interest and is constitutively degraded by the proteosome. Addition of the ligand trimethoprim (TMP) can stabilize DHFR(DD), preventing proteasomal degradation of the fusion protein and thereby inducing accumulation in cells. We synthesized and optimized TRimethoprim Analog Probes that maintain stabilization activity and contain a terminal alkyne for Click functionalization and a thiol reactive group to covalently tag DHFR(DD). Click reaction with a biotin tag and subsequent streptavidin enrichment enable time-resolved mass spectrometric identification of interacting partners. We evaluated the timing of protein interactions of SARS-CoV-2 and SARS-CoV nonstructural protein 15 (nsp15) over a 2 h period. We found interactors GEMIN5 and YBX3, known regulators of SARS-CoV-2 infection that bind viral RNA, as well as CACYBP and FHL1 that implicate nsp15 in the disruption of host ERK1/2 signaling. We further revealed that these interactions remain relatively steady from 0 to 2 h post translation of nsp15. TRAPPED methodology can be applied to determine the sequence and timing of protein-protein interactions of temporally regulated biological processes such as viral infection or signal transduction.