Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function

Area of Science:

• Molecular Biology
• Neuroscience
• Genetics

Background:

• Mutations in the TBC1D24 gene are linked to severe epilepsy and DOORS syndrome.
• The precise molecular mechanisms driving these TBC1D24-associated pathologies remain unclear.

Purpose of the Study:

• To elucidate the molecular function of TBC1D24 and the impact of disease-associated mutations.
• To investigate the role of phosphoinositide binding in TBC1D24 presynaptic function.

Main Methods:

• Determined the crystal structure of the Drosophila TBC1D24 ortholog, Skywalker.
• Performed cocrystallization and biochemical assays to identify phosphoinositide binding.
• Utilized in vivo photobleaching and genetic manipulation in Drosophila models to assess protein function and rescue phenotypes.

Main Results:

• The TBC domain of Skywalker possesses a conserved cationic pocket that binds phosphoinositides (specifically those phosphorylated at the 4 and 5 positions).
• Common patient mutations in TBC1D24 disrupt this pocket, inhibiting phosphoinositide binding and Skywalker's membrane association.
• Pathogenic mutations lead to impaired synaptic vesicle trafficking and seizures in flies, which are ameliorated by increasing PI(4,5)P2 levels.

Conclusions:

• TBC1D24 directly binds phosphoinositides via a cationic pocket in its TBC domain.
• Phosphoinositide binding is essential for TBC1D24's role in presynaptic function.
• Understanding this mechanism provides insights into TBC1D24-related epilepsy and DOORS syndrome.