Time-resolved cryo-EM (TR-EM) analysis of substrate polyubiquitination by the RING E3 anaphase-promoting...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Substrate polyubiquitination is essential for key cellular processes like cell cycle regulation, apoptosis, and immune responses.
• Understanding the dynamic mechanisms of polyubiquitination has been challenging due to the transient nature of intermediates, often requiring artificial stabilization.
• The precise mechanism by which E3 ubiquitin ligases assemble polyubiquitin chains, particularly the canonical four or more ubiquitins signaling for proteasomal degradation, remains largely unknown.

Purpose of the Study:

• To elucidate the dynamic mechanism of substrate polyubiquitination by the anaphase-promoting complex/cyclosome (APC/C).
• To visualize the direct interactions between the E3 ligase, E2 co-enzymes, and substrates during ubiquitin chain formation.
• To model how E3 ligases, specifically the APC/C, construct proteasomal degradation signals.

Main Methods:

• Time-resolved cryogenic electron microscopy (cryo-EM) was employed to capture snapshots of the APC/C during polyubiquitination.
• CryoDRGN, a neural network-based approach, was utilized to reconstruct conformational dynamics from the cryo-EM data.
• The study focused on the human APC/C complex with its cognate E2 co-enzymes, UBE2C/UBCH10 and UBE2S.

Main Results:

• Reconstruction of the conformational landscape of the human APC/C during the polyubiquitination process.
• Direct visualization of an active E3-E2 enzyme pair modifying a substrate.
• Identification of novel interactions between nascent ubiquitin chains and the APC/C machinery, including its coactivator CDH1.
• Demonstration that modification with nascent ubiquitin chains enhances processive polyubiquitination.

Conclusions:

• The study provides unprecedented mechanistic insight into how the APC/C builds polyubiquitin chains.
• A model is proposed for how ubiquitin ligases generate proteasomal degradation signals through potentiated polyubiquitination.
• These findings advance our understanding of ubiquitin signaling and its role in fundamental cellular processes.