Biophysical Analysis of CRBN Variants in PROTAC Ternary Kinetics

Proteolysis-targeting chimeras (PROTACs) and molecular glues have emerged as powerful therapeutic modalities by exploiting the ubiquitin–proteasome system to drive selective protein degradation. However, structural and biophysical characterization of these degraders is often hampered by the complexity of full-length E3 ligases, particularly cereblon (CRBN), one of the most extensively studied and clinically relevant ligases. CRBNmidi, a truncated CRBN construct, overcomes these limitations and enables detailed analysis of analysis of degrader-induced ternary complex formation and stability. Here, we compare PROTAC-mediated ternary complexes formed with CRBNmidi or the full-length DDB1:CRBN complex in the presence of Brd4 using the switchSENSE^®
Y-structure binding assay. Our results demonstrate that CRBNmidi forms more stable ternary complexes with slower dissociation kinetics than the full-length E3 ligase. This effect likely arises from the absence of DDB1, which leaves the sensor loop unbound, destabilizes the open state, and favors the closed, ligand-bound conformation. Together, these findings establish CRBNmidi as a valuable tool for mechanistic studies and highlight the utility of the DNA Y-structure assay for the characterization and optimization of protein degraders.