Improved Stability of Human CGI-58 Induced by Phosphomimetic S237E Mutation

Area of Science:

• Biochemistry
• Molecular Biology
• Cellular Metabolism

Background:

• CGI-58's lipolytic activity is regulated by its interaction with perilipin 1 (PLIN1) on lipid droplets.
• Phosphorylation controls CGI-58 release, enabling recruitment of lipases like ATGL and HSL for fatty acid release.
• Mouse CGI-58 S239E mutant mimics phosphorylation, dissociates from PLIN1, and activates ATGL.

Purpose of the Study:

• To analyze the stabilizing effects of mutations on human CGI-58.
• To investigate the impact of a triple tryptophan to alanine (3WA) mutant on the LD-binding motif.
• To examine a quadruple mutant (3WA/S237E) incorporating a phosphomimetic substitution.

Main Methods:

• Site-directed mutagenesis to create 3WA and 3WA/S237E CGI-58 variants.
• Assessing protein aggregation, thermal stability, and solubility.
• Evaluating protein folding and binding affinity for oleoyl-CoA.
• Bioinformatic analysis using a 3D model.

Main Results:

• Tryptophan residues in WT CGI-58 promote protein aggregation; alanine substitution favors monomeric form.
• The 3WA/S237E mutant exhibited enhanced thermal stability and solubility compared to the 3WA mutant.
• 3WA/S237E protein demonstrated proper folding and a functional oleoyl-CoA binding site.
• Bioinformatic modeling suggested an intramolecular interaction in 3WA/S237E, potentially explaining its stability.

Conclusions:

• Mutations altering tryptophan residues and mimicking serine 237 phosphorylation improve CGI-58 solubility and stability.
• The findings support a role for serine 237 phosphorylation in regulating CGI-58 function.
• These insights contribute to understanding lipolysis regulation at the molecular level.