Unmasking Crucial Residues in Adipose Triglyceride Lipase (ATGL) for Co-Activation with Comparative Gene Identification-58 (CGI-58)

Lipolysis is an essential metabolic process that releases unesterified fatty acids from neutral lipid stores to maintain energy homeostasis in living organisms. Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis and can be co-activated upon interaction with the protein comparative gene identification-58 (CGI-58). The underlying molecular mechanism of ATGL/CGI-58 stimulation is incompletely understood. Based on analysis of evolutionary conservation we used site directed mutagenesis to study a C-terminally truncated variant and full-length mouse ATGL providing insights in the protein co-activation on a per-residue level. We identified the region from residues N209-N215 in mouse ATGL as essential for co-activation by mouse CGI-58. ATGL variants with amino-acids exchanges in this region were still able to hydrolyze triacylglycerol at the basal level and to interact with CGI-58, yet could not be co-activated by CGI-58. Our studies also demonstrate that full-length mouse ATGL showed higher tolerance to specific single amino acid exchanges in the N209-N215 region upon CGI-58 co-activation. The region is either directly involved in protein-protein interaction or essential for conformational changes required in the co-activation process. We generated three-dimensional models of the ATGL/CGI-58 complex with the artificial intelligence software AlphaFold demonstrating that a large surface area is involved in the protein-protein interaction. The model is in very good agreement with the herein presented data identifying important residues of ATGL and published data revealing essential residues of CGI-58 for co-activation. ### Competing Interest Statement The authors have declared no competing interest.