Mechanism of degrader-targeted protein ubiquitinability

Small molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and the potential to tackle previously undrugged targets. Thermodynamically stable and kinetically long-lived degrader-mediated ternary complexes can drive faster, more profound and durable target degradation, however the mechanistic features by which they impact on target ubiquitination remain elusive. Here, we solve cryo-EM structures of the VHL Cullin 2 RING E3 ligase complexed with degrader MZ1, target protein Brd4BD2 and primed for catalysis with its cognate E2-ubiquitin bound. We find that Brd4BD2 adopts a favourable orientation towards the E2 active site. In vitro ubiquitination coupled with mass spectrometry illuminates a patch of ubiquitinable lysines on one face of Brd4BD2, with Lys456 showing optimal distance and geometry for nucleophilic attack. Our results demonstrate the proficiency of MZ1 in directing the substrate towards catalysis, explains the favourability of Brd4BD2 for ubiquitination, and reveals the flexibility of the enzyme in capturing sub-optimal lysines. We propose a model for ubiquitinability of degrader-recruited targets that provides a mechanistic blueprint for further rational drug design and optimization. ### Competing Interest Statement A.C. is a scientific founder, shareholder and advisor of Amphista Therapeutics, a company that is developing targeted protein degradation therapeutic platforms. The Ciulli laboratory receives or has received sponsored research support from Almirall, Amgen, Amphista Therapeutics, Boehringer Ingelheim, Eisai, Merck KaaG, Nurix Therapeutics, Ono Pharmaceutical and Tocris-Biotechne. The other authors declare no competing interests.