Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) alter mitochondrial morphology and result in several subtypes of the inherited peripheral neuropathy Charcot-Marie-Tooth disease; however, the mechanism by which GDAP1 functions has remained elusive. GDAP1 contains primary sequence homology to the GST superfamily; however, the question of whether GDAP1 is an active GST has not been clearly resolved. Here, we present biochemical evidence, suggesting that GDAP1 has lost the ability to bind glutathione without a loss of substrate binding activity. We have revealed that the α-loop, located within the H-site motif is the primary determinant for substrate binding. Using structural data of GDAP1, we have found that critical residues and configurations in the G-site which canonically interact with glutathione are altered in GDAP1, rendering it incapable of binding glutathione. Last, we have found that the overexpression of GDAP1 in HeLa cells results in a mitochondrial phenotype which is distinct from oxidative stress-induced mitochondrial fragmentation. This phenotype is dependent on the presence of the transmembrane domain, as well as a unique hydrophobic domain that is not found in canonical GSTs. Together, we data point toward a non-enzymatic role for GDAP1, such as a sensor or receptor. K E Y W O R D Sganglioside-induced differentiation-associated protein 1, mitochondria, oxidative stress, structural biology, X-ray crystallography

GDAP1 (Ganglioside-induced differentiation-associated protein 1) is a novel member of the GST superfamily of detoxifying enzymes that is anchored to the outer mitochondrial membrane. GDAP1 mutations and changes in expression levels result in the inherited neuropathy Charcot-Marie-Tooth (CMT) disease, types 2K, 4A and 4H. GDAP1 activity has been associated with many mitochondrial functions however direct molecular interactions underpinning these connections have remained elusive. Here we establish that GDAP1 can bind 4-hydroxynonenal (4HNE), a toxic end-product of lipid peroxidation. 4HNE binding requires the α-loop, a large sequence motif that is inserted within the substrate recognition domain and is unique to GDAP1. In human cells, GDAP1 overexpression plays a cytoprotective role against oxidative stress. This effect is lost upon deletion of the alpha loop. Lastly, we demonstrate that a CMT-causing mutant that destabilizes alpha loop positioning also results in a decrease in 4HNE binding affinity. Together these results establish 4HNE as the biological ligand for GDAP1, provide mechanistic insight into 4HNE binding, and demonstrate that altered 4HNE recognition is the likely mechanism underlying CMT-causing mutants such as T157P near the 4HNE binding site.