Deep mutational interaction perturbation of ATGL

ATGL is the key enzyme in intracellular lipolysis impacting energy metabolism, glucose tolerance, insulin sensitivity, thermogenesis and has a critical role in several metabolic and cardiovascular diseases. ATGL is regulated through a defined set of protein-protein interaction partners with tissue specific activating and inhibiting functions. However, the binding mode and protein interaction sites of ATGL with its essential interaction partners are unknown hampering a deeper mechanistic understanding of the molecular events impacting lipolysis. Here we used deep mutational protein interaction perturbation scanning to generate comprehensive profiles of single amino acid variants effecting the interactions of ATGL with its most important regulatory partners, CGI-58, G0S2, Plin1, Plin5 and CIDEC. Twenty-three variants gave a specific ATGL interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We elucidated and characterized twelve ATGL “switch” mutations which selectively affect the interaction of one of the five partners with no effect on the others. Switch mutations provided distinct interaction determinants for ATGL's key regulatory proteins at amino acid resolution. When tested for triglyceride hydrolase activity, the activity patterns of the ATGL switch variants exactly matched the protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles allowed important insight into lipolysis mechanism and the impact of mutations in human disease.