LC3B Binds to the Autophagy Protease ATG4b with High Affinity Using a Bipartite Interface

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Autophagy is a vital cellular process for degrading waste proteins and organelles using lysosomes.
• LC3B protein is key in autophagosome formation and fusion with lysosomes.
• The cysteine protease ATG4b regulates LC3B function at multiple stages.

Purpose of the Study:

• To elucidate the protein-protein interaction (PPI) between LC3B and ATG4b.
• To characterize the binding kinetics and inhibitory potential of LC3B variants with ATG4b.
• To understand the structural basis of LC3B-ATG4b recognition and catalysis.

Main Methods:

• Biochemical assays to measure binding affinity (K_D) and inhibition (IC₅₀).
• Biophysical studies to assess protein-protein interactions.
• Site-directed mutagenesis to analyze the role of protein domains and tails.

Main Results:

• The ubiquitin-like core of LC3B (LC3B-115) binds ATG4b with high affinity (low nanomolar K_D), 10-30x tighter than its substrate or product.
• LC3B-115 is a potent inhibitor of ATG4b, with an IC₅₀ of 15 nM.
• ATG4b's C-terminal tail is essential for high-affinity LC3B binding and proteolysis, while LC3B's C-terminal tail organizes the ATG4b active site.

Conclusions:

• A bipartite model for LC3B-ATG4b interaction is proposed, involving core LC3B binding and C-terminal pro-LC3B-mediated active site organization.
• The C-terminal tail of ATG4b significantly enhances binding affinity (>1000-fold) and likely interacts with the LC3B-ubiquitin core.
• The LC3B-ATG4b complex features multiple energetically significant domains, influencing both binding and catalytic efficiency in autophagy regulation.