Structure and mechanism of the HECT ligase HECTD3

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• HECT E3 ligases are crucial regulators of cellular processes, but their substrate recognition and poly-ubiquitin chain synthesis mechanisms remain unclear.
• HECTD3, an "other HECT" family member, plays roles in inflammation, apoptosis, infection, and is highly expressed in cancers, primarily due to its ligase activity.

Purpose of the Study:

• To comprehensively analyze the ligase activity of HECTD3, including ubiquitin linkage preferences, oligomeric state, and substrate ubiquitination.
• To elucidate the full-length structures of HECTD3 in apo and ubiquitin-loaded states using cryo-electron microscopy (cryo-EM).

Main Methods:

• Detailed biochemical analysis of HECTD3 ligase activity.
• Cryo-electron microscopy (cryo-EM) for full-length HECTD3 structural determination.

Main Results:

• The study provides full-length HECTD3 structures in apo and ubiquitin-loaded forms, offering insights into its domain organization.
• A distinct N-terminal region fold was discovered, alongside mechanistic features shared with other HECT ligases and unique to HECTD3.
• HECTD3 exhibits specific ubiquitin linkage preferences and modifies diverse substrates.

Conclusions:

• The structural and mechanistic insights into HECTD3 explain its functional diversity and catalytic differences compared to other HECT ligases.
• Understanding HECTD3's unique features can inform its role in cancer and other diseases.