Autophagy is a crucial cellular catabolic process that degrades unwanted cytoplasmic material (cargo) by engulfing the material into a double-membrane vesicle (autophagosome) and then presenting it to the lysosome. The work in this thesis explores interactions between the proteins involved in the autophagy pathway.

Focal adhesion complex Interacting Protein of 200 kDa (FIP200) and autophagy related protein 13 (Atg13) are members of the autophagy initiation complex. Attempts were made to locate the region on FIP200 that binds the Atg13 by dividing FIP200 into six fragments of equal length and performing pull-down assays with Atg13. However, because of a false positive interaction observed for all six FIP200 fragments, binding between the Atg13- interaction region on FIP200 and Atg13 could not be identified.

Pleckstrin homology domain-containing protein family member 1 (PLEKHM1) is a selective autophagy receptor that interacts with the autophagy-related protein 8 homologues (Atg8 proteins: LC3A, LC3B, LC3C and GABARAP, GABARAPL1, GABARAPL2). The affinity of PLEKHM1 for the LC3 family is higher than that for the GABARAP family of proteins. The Atg8 proteins are involved in tethering cargo to the autophagosome. The crystal structures of five human ATG8 proteins were solved in complex with a peptide with the sequence of the PLEKHM1 LC3-Interacting Region (LIR) to understand the structural basis of their differing binding affinities. These structures demonstrated that residues Arg28 and Glu17 of the GABARAP family proteins interact with Asn637 and Trp635 residues of the PLEKHM1 LIR peptide. However, these interactions were either weak or not present in the LC3 family proteins, suggesting that they may contribute to the higher binding affinity for the PLEKHM1 LIR observed with GABARAP proteins. In addition, a structural comparison of the human ATG8 proteins suggested that the “W-site” hydrophobic pocket, where Trp635 in PLEKHM1 binds, is deeper in GABARAP family proteins than in LC3 family proteins. Together, the structural analysis provided a rationale for varying binding affinities between GABARAP and LC3 family proteins.

The serine/threonine-protein kinase ULK1, which is part of the autophagy initiation complex, also interacts with the Atg8 family proteins. Using surface plasmon resonance, molecular dynamic (MD) simulations, mutational analysis and X-ray crystallography, the interaction of the ULK1 LIR with GABARAP and LC3A was studied to understand the difference in the LIR interaction mechanism. Structural comparison of the LC3A and GABARAP proteins in their LIR-bound and unbound states showed that LC3A shows more variation in the structure of its “W-site” hydrophobic pocket between its bound and unbound states than the “W-site” of GABARAP. This was verified by MD simulations. Additionally, the MD simulations suggested an electrostatic interaction between LC3A Arg70 and ULK1 Met359. This was demonstrated by the decrease in binding affinity when Arg70 was mutated to leucine. In contrast, mutation of Arg67 (which is equivalent to Arg70 in LC3A) to leucine did not have any effect on LIR binding.

Overall, this work provides key insights into the differences in the mechanisms of interaction demonstrated by the GABARAP and LC3 family proteins, which is an initial step towards understanding the different functions of the members of the two human ATG8
subfamilies (GABARAP and LC3).