Characterization of ATG8-Family Interactors by Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) is the gold standard for providing quantitative and thermodynamic understanding of the interaction mechanisms between core autophagy machinery, autophagy receptors, and ATG8. Here, we used two model peptides and Arabidopsis thaliana ATG8A to characterize ATG8–peptide interactions. We employed ITC using three different methods (direct ligand titration, displacement, and competition assays) to characterize, directly and indirectly, the interaction of the peptides with ATG8. We then analyzed the ITC data by global and statistical methods and discussed advantages, drawbacks, and negative controls for each approach. We finally provide a thorough description of all the steps, including data analysis and presentation, preparation of recombinant ATG8A from E. coli, and troubleshooting notes for technical problems that can be encountered. Although we used ATG8–peptide interactions here, these assays can be applied to any other one-to-one protein–protein and ligand–protein interactions and competitive binders.