A pathogenic mutation in α-SNAP impairs membrane lipid binding by concealing a critical hydrophobic loop

The soluble N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP) is essential for vesicle trafficking, coordinating trans-SNARE zippering and cis-SNARE disassembly. α-SNAP also regulates autophagy, apoptosis, calcium signaling, and AMPK activity. The hyh missense mutation M105I produces a distinctive neurodevelopmental phenotype, yet its pathogenic mechanism remains unclear. Because many α-SNAP functions rely on lipid binding, we examined whether M105I alters this property. In silico modeling revealed structural rearrangements that conceal the N-terminal hydrophobic loop, and molecular dynamics simulations predicted reduced binding free energy and weakened protein–lipid interactions. These predictions were validated in vitro and in hyh mouse brains, showing diminished membrane association, particularly at the plasma membrane. Liposome flotation assays with plasma-membrane–derived lipids confirmed that M105I directly impairs lipid binding and that membrane composition influences this interaction. Thus, defective lipid engagement emerges as a central determinant of α-SNAP dysfunction and likely contributes to the pathogenesis of hyh phenotype. (Figure presented.)