Macroautophagy is one of two major degradation systems
in eukaryotic
cells. Regulation and control of autophagy are often achieved through
the presence of short peptide sequences called LC3 interacting regions
(LIR) in autophagy-involved proteins. Using a combination of new protein-derived
activity-based probes prepared from recombinant LC3 proteins, along
with protein modeling and X-ray crystallography of the ATG3-LIR peptide
complex, we identified a noncanonical LIR motif in the human E2 enzyme
responsible for LC3 lipidation, ATG3. The LIR motif is present in
the flexible region of ATG3 and adopts an uncommon β-sheet structure
binding to the backside of LC3. We show that the β-sheet conformation
is crucial for its interaction with LC3 and used this insight to design
synthetic macrocyclic peptide-binders to ATG3. CRISPR-enabledin cellulostudies provide evidence that LIRATG3is required for LC3 lipidation and ATG3∼LC3 thioester formation.
Removal of LIRATG3negatively impacts the rate of thioester
transfer from ATG7 to ATG3.