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

Area of Science:

• Molecular Biology
• Neuroscience
• Biochemistry

Background:

• Soluble N-ethylmaleimide-sensitive factor attachment protein alpha (α-SNAP) is crucial for vesicle trafficking and cellular signaling.
• The hyh missense mutation M105I causes a neurodevelopmental phenotype, but its underlying mechanism is unknown.
• α-SNAP's functions are known to involve lipid binding.

Purpose of the Study:

• To investigate whether the hyh mutation M105I affects the lipid-binding properties of α-SNAP.
• To elucidate the pathogenic mechanism of the hyh phenotype by examining α-SNAP's interaction with membranes.

Main Methods:

• In silico modeling and molecular dynamics simulations to predict structural changes and binding affinities.
• In vitro experiments, including liposome flotation assays, to validate predicted lipid-binding defects.
• Analysis of α-SNAP membrane association in hyh mouse brains.

Main Results:

• In silico analysis predicted that M105I causes structural rearrangements that reduce α-SNAP's lipid binding.
• Experimental validation confirmed diminished membrane association of M105I α-SNAP, especially at the plasma membrane.
• Liposome assays demonstrated that the M105I mutation directly impairs α-SNAP's lipid binding, with effects modulated by membrane composition.

Conclusions:

• Defective lipid engagement by α-SNAP due to the M105I mutation is a primary cause of its dysfunction.
• Impaired α-SNAP lipid binding likely contributes significantly to the pathogenesis of the hyh neurodevelopmental phenotype.