Structural Insights into the GABARAP-ATG3 Backside Interaction and Apo ATG3 Conformation

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Autophagy is a vital cellular process for degrading damaged components.
• ATG8 proteins, like GABARAP, are essential for autophagosome formation.
• The lipidation pathway of ATG8 proteins, particularly the role of ATG3, requires further molecular elucidation.

Purpose of the Study:

• To investigate the molecular mechanisms of ATG8 protein lipidation by the E2 enzyme ATG3.
• To uncover novel binding interactions between GABARAP and ATG3.
• To understand the regulatory mechanisms governing ATG3 activity.

Main Methods:

• X-ray crystallography to determine the structure of GABARAP-ATG3 conjugates.
• Solution Nuclear Magnetic Resonance (NMR) spectroscopy to confirm interactions.
• Site-directed mutagenesis to assess the functional importance of the identified interface.
• AlphaFold modeling to predict the structure of apo ATG3.

Main Results:

• A novel noncovalent binding mode between GABARAP and the backside of ATG3's catalytic domain was identified.
• Crystallographic analysis revealed self-assembly into a helical filament via this backside interface.
• NMR and mutagenesis confirmed the interaction's role in phosphatidylethanolamine (PE) conjugation.
• Intramolecular contacts within ATG3 were found to suppress conjugation.

Conclusions:

• Backside engagement represents a critical, previously unrecognized feature of ATG8 lipidation.
• The findings illuminate the dynamic architecture and regulatory mechanisms of ATG3.
• This study provides new insights into the fundamental process of autophagy.