AMBRA1 is a critical tumor suppressor protein acting as a substrate receptor within the ubiquitin conjugation system, with established roles in autophagy and cell-cycle regulation. Its intrinsic disorder has limited structural characterization. To overcome this, we employed hydrogen deuterium exchange mass spectrometry (HDX-MS) and cryo-electron microscopy (cryo-EM) to probe the architecture and dynamics of AMBRA1 and its regulatory complexes. HDX-MS revealed AMBRA1 to be highly flexible, stabilizing upon association with DDB1, an adaptor of the Cullin4A/B E3 ligase. Our cryo-EM structures resolved both the AMBRA1-DDB1 complex at 3.08 Å and the cyclin D1-bound AMBRA1-DDB1 complex at 3.55 Å, uncovering how AMBRA1’s WD40 domain coordinates substrate binding and recruitment. Notably, interaction with the phosphorylated Thr286 residue within the cyclin D1 phosphodegron is essential for substrate recognition, with mutants disrupting these engagements leading to cyclin D1 accumulation and DNA damage. AMBRA1 acts as a molecular hub, integrating signals through its dynamic interactions with DDB1 and D-type cyclins to orchestrate substrate recognition and cell-cycle progression. Collectively, our findings provide a high-resolution molecular framework for AMBRA1-dependent ubiquitin ligase activity and cell-cycle control, offering new mechanistic insights and potential therapeutic strategies for cancers involving dysregulated cyclin D activity.
Goran Stjepanovic (Sun,) studied this question.