Biochemical and Structural Characterization of the Atg8/LC3 Lipidation Pathway

Atg8 and its eukaryotic orthologues LC3 and GARBARAP family proteins (referred here to Atg8 family proteins) play crucial roles in autophagy through their covalent ligation to lipids, typically phosphatidylethanolamine (PE), in a process known as lipidation. Lipidation of Atg8 family proteins regulates numerous facets of the autophagy process, including regulating expansion of the phagophore membrane, recruiting selected cargoes for degradation, and providing an autophagosome membrane-bound platform mediating dynamic interactions with other regulatory proteins. Atg8 family proteins are ubiquitin-like proteins (UBLs), and their lipidation involves a divergent UBL conjugation cascade including Atg7, Atg3, and Atg12–Atg5-Atg16 acting as E1, E2, and E3 enzymes, respectively. Atg7 initiates Atg8 conjugation by catalyzing their C-terminal adenylation and conjugation to the catalytic cysteine of Atg3. Ultimately, the Atg12–Atg5-Atg16 complex catalyzes Atg8 ligation to a primary amino group on PE or other acceptor lipids. Molecular mechanisms underlying Atg8 lipidation remain poorly understood despite association of Atg3, the E1 Atg7, and the composite E3 Atg12–Atg5-Atg16 with pathologies including cancers, infections, and neurodegeneration. The first part of this dissertation work describes methods for expressing and purifying human LC3 or GABARAP, ATG7, ATG3, and the ATG12–ATG5-ATG16L1 complex for in vitro studies of LC3/GABARAP lipidation; based on these protocol established, we report that an Atg3 element we term E123IR (E1, E2, and E3-interacting region) is an allosteric switch, by studying yeast enzymes. Nuclear magnetic resonance (NMR), biochemical, crystallographic and genetic data collectively indicate that in the absence of the enzymatic cascade, the Atg3E123IR makes intramolecular interactions restraining Atg3’s catalytic loop, while E1 and E3 enzymes directly remove this brace to conformationally activate Atg3 and elicit Atg8 lipidation in vitro and in vivo. We propose that Atg3’s E123IR protects the E2~UBL thioester bond from wayward reactivity toward errant nucleophiles, while Atg8 lipidation casca