Dual E3 ligase recruitment by monovalent degraders enables redundant and tuneable degradation of SMARCA2/4
Proteolysis-Targeting Chimeras (PROTACs) and Molecular Glue Degraders (MGDs) canonically target proteins for degradation by recruiting them to a single E3 ligase complex. While heterotrivalent PROTACs that can co-opt multiple E3 ligase complexes have been described, to our knowledge all MGDs reported to date are dependent on a single E3. Here, using orthogonal genetic screening, biophysical and structural analyses, we show that a monovalent MGD can covalently recruit CUL4DCAF16 and CRL1FBXO22 in a parallel and redundant manner to degrade SMARCA2/4. Deep mutational scanning identifies a single cysteine (Cys173) in DCAF16 essential for degrader activity, and intact protein MS confirms covalent adduct at this site. The cryo-EM structure of the DCAF16:SMARCA2:degrader ternary complex reveals a unique binding mode and a distinct interface of neo-interactions, providing insights into degrader specificity. We demonstrate that E3 ligase dependency can be tuned both chemically and genetically. Minimal alterations to the compounds "degradation tail" switches ligase preference from DCAF16 to FBXO22, while a single L59W mutation on DCAF16 is sufficient to drive DCAF16 engagement for otherwise FBXO22-dependent compounds. These results establish a molecular and structural framework for the design of tuneable dual glue degraders that could mitigate challenges from resistance mechanisms in degrader therapies.