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.