Fig. 1: Mapping ATGL protein interaction determinants using deep mutational interaction perturbation scanning.

A Schematic overview of ATGL and its interaction partners. Known AA residues mediating the interactions are annotated, see text for references. The Perilipin family members bind ATGL within the patatin domain. G0S2 also binds ATGL within the patatin domain, inhibits its hydrolytic activity. CIDEC, another lipolysis inhibiting protein interacts with the C-terminal part of ATGL. CGI-58 binds ATGL via the patatin domain as well as the C-terminal part. However, no interactions sites on ATGL for its partners were identified. B Schematic overview of the deep mutational interaction perturbation screening approach. Plasmid libraries of ATGL were generated using array programmed deep mutagenesis, exchanging single amino acid to A, K, E, or L, respectively. Reverse Y2H strains were transformed with the ATGL libraries and mated with yeast strains expressing a WT interaction partner. Interaction-disrupting mutations were enriched through yeast growth, and mutations perturbing the PPI were identified by NGS sequencing. C Scatter plot of read counts of library 1 in the Gateway Entry vector after deep mutagenesis with the library after yeast mating prior to growth selection. Read counts compared for sum of mutants per codon (left) as well as per position (right). R2 values indicate that the library did not change during the cloning procedure. D Scatter plot of read counts of the two independent ATGL libraries. Library 1 and Library 2 were compared for their sum of mutants observed per codon (left) as well as per position (right). Red colored circles indicate programmed AKEL mutations which are higher in library 2 than library 1. The outlier data point (Position 1) in library 1 in the right graph likely stems from an PCR amplification step early during preparation.