Tuning the open-close equilibrium of Cereblon with small molecules influences protein degradation.

Most PROTACs and molecular glue degraders currently approved or in clinical trials recruit Cereblon (CRBN) as the ubiquitin E3 ligase. Upon binding ligands and molecular glues, CRBN undergoes a significant structural rearrangement from an open to closed state, defined by the positioning of the thalidomide-binding domain (TBD) with respect to the Lon domain. However, the exact molecular basis for this ligand-induced conformational change and its implication to neo-substrate degradation remain elusive. During our campaign to discover novel CRBN binders, we found hits exhibiting distinct biophysical behaviour from classical thalidomide-based ligands. By combining orthogonal biophysical methods of differential scanning fluorimetry, isothermal titration calorimetry, and small-angle X-ray scattering, supported by X-ray crystallography and cryo-EM structures of ligand-bound complexes, we classify CRBN binders between those that can induce CRBN closure, and those that cannot. Mutational studies identify key residues in the CRBN ligand binding pocket and N-terminal belt that are essential for the Lon and TBD domains to come together in the closed state. Finally, we show that the probability to yield active degrader molecules is greatly influenced by whether binders can or cannot induce CRBN closure. Together, our study reveals new molecular insights into the structural basis for how CRBN open-closed equilibrium is directly modulated by compound binding and impact target degradability by CRBN, with important implications to the design of PROTACs and molecular glue degraders.