Structure of a CGI-58 Motif Provides the Molecular Basis of Lipid Droplet Anchoring

Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10–31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp²¹ and Trp²⁵) and right (harboring Trp²⁹) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp²¹ and Trp²⁵ and two adjacent leucines. Trp²⁹ serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm.