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

[Image: see text] Autophagy is a catabolic cellular process in which unwanted proteins and organelles are degraded by lysosomes. It is characterized by the formation of the double-membrane autophagosome decorated with LC3B, a protein that mediates autophagosomal fusion with lysosomes. The cysteine protease ATG4b acts at two stages in the life cycle of LC3B. We set out to characterize the protein–protein interaction between LC3B and ATG4b. Through biochemical and biophysical studies, we show that the ubiquitin-like core of LC3B (residues 1–115; “LC3B-115”), which lacks the C-terminal cleavage site (between residue 120 and 121), binds to full-length ATG4b with a surprisingly tight dissociation constant (K(D)) in the low nanomolar range; 10–30-fold tighter than that of the substrate pro-LC3B (residues 1–125) or the product LC3B-I (residues 1–120). Consequently, LC3B-115 is a potent inhibitor of the ATG4b-mediated cleavage of pro-LC3B (IC(50) = 15 nM). Binding of the LC3B-115 has no effect on the conformation of the active site of ATG4b, as judged by the turnover of a peptide substrate (“substrate-33”), derived from LC3B-I residues 116–120. Conversely, truncations of ATG4b show that binding and proteolysis of LC3B critically depend on the C-terminal tail of ATG4b, whereas proteolysis of the peptide substrate-33 does not require the C-terminal tail of ATG4b. These results support a bipartite model for LC3B-ATG4b binding in which the core of LC3B binds to ATG4b and the C-terminal tail of pro-LC3B organizes the ATG4b active site; additionally, the C-terminal tail of ATG4b contributes at least 1000-fold higher binding affinity to the LC3B-ATG4b interaction and likely wraps around the LC3B-ubiquitin core. PPIs are often described as containing an energetic “hot spot” for binding; in the case of LC3B-ATG4b, however, the substrate–enzyme complex contains multiple, energetically relevant domains that differentially affect binding affinity and catalytic efficiency.