Members of the carboxylesterase 2 (Ces2/CES2) family have been studied intensively with respect to their hydrolytic function on (pro)drugs, whereas their physiological role in lipid and energy metabolism has been realized only within the last few years. Humans have one CES2 gene which is highly expressed in liver, intestine, and kidney. Interestingly, eight homologous Ces2 (Ces2a to Ces2h) genes exist in mice and the individual roles of the corresponding proteins are incompletely understood. Mouse Ces2c (mCes2c) is suggested as potential ortholog of human CES2. Therefore, we aimed at its structural and biophysical characterization. Here, we present the first crystal structure of mCes2c to 2.12 Å resolution. The overall structure of mCes2c resembles that of the human CES1 (hCES1). The core domain adopts an α/β hydrolase-fold with S230, E347, and H459 forming a catalytic triad. Access to the active site is restricted by the cap, the flexible lid, and the regulatory domain. The conserved gate (M417) and switch (F418) residues might have a function in product release similar as suggested for hCES1. Biophysical characterization confirms that mCes2c is a monomer in solution. Thus, this study broadens our understanding of the mammalian carboxylesterase family and assists in delineating the similarities and differences of the different family members.

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.