Conformational plasticity in GDAP1's GST-like domain enables thapsic acid recognition

The GST superfamily of proteins canonically use a large central active site to recognize and detoxify the cell from a range of damaged lipids and xenobiotics. The mitochondrial GST-like protein, GDAP1, was proposed to bind the dicarboxylic fatty acid, thapsic acid, through a non-canonical binding pocket, similar to a GST L-site, allowing GDAP1 to adopt an unusually open conformation, and leaving the function of the canonical active site and an adjacent feature called the alpha-loop unclear. Here, we report the biochemical impact of mutants in all of GDAP1's canonical domains (G-site, H-site, alpha-loop, and putative L-site), revealing that thapsic acid binding activity is actually housed within the canonical active site pocket. Our biochemistry, combined with structural predictions and modeling strongly suggests that movements in the alpha-loop and G-site effectively remodel the canonical GDAP1-binding pocket to facilitate interactions with thapsic acid. Further, mutants in the canonical active site eliminated the cytoprotective effects of wild-type GDAP1. Together, these findings establish GDAP1 as a conformationally dynamic GST-like protein in which coordinated alpha-loop and G-site movements remodel the canonical active site to enable lipid recognition, coupling its biochemical activity to mitochondrial membrane integrity and redox homeostasis. Furthermore, we provide an extensive map of residues that coordinate lipid binding by GDAP1.