GDAP1 binds 4-hydroxynonenal, the toxic end-product of lipid peroxidation, using its GST-like binding pocket

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 -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 -loop. Lastly, we demonstrate that a CMT-causing mutant that destabilizes -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.