Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function.
Mutations in TBC1D24 cause severe epilepsy and DOORS syndrome, but the molecular mechanisms underlying these pathologies are unresolved. We solved the crystal structure of the TBC domain of the Drosophila ortholog Skywalker, revealing an unanticipated cationic pocket conserved among TBC1D24 homologs. Cocrystallization and biochemistry showed that this pocket binds phosphoinositides phosphorylated at the 4 and 5 positions. The most prevalent patient mutations affect the phosphoinositide-binding pocket and inhibit lipid binding. Using in vivo photobleaching of Skywalker-GFP mutants, including pathogenic mutants, we showed that membrane binding via this pocket restricts Skywalker diffusion in presynaptic terminals. Additionally, the pathogenic mutations cause severe neurological defects in flies, including impaired synaptic-vesicle trafficking and seizures, and these defects are reversed by genetically increasing synaptic PI(4,5)P2concentrations through synaptojanin mutations. Hence, we discovered that a TBC domain affected by clinical mutations directly binds phosphoinositides through a cationic pocket and that phosphoinositide binding is critical for presynaptic function.
Structural Biology Research Center, VIB, Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Baptiste
B
Fischer
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Kevin
http://orcid.org/0000-0002-4510-3826
K
Lüthy
Structural Biology Research Center, VIB, Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Jone
J
Paesmans
Structural Biology Research Center, VIB, Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Charlotte
C
De Koninck
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Ine
I
Maes
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Jef
J
Swerts
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Sabine
S
Kuenen
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Valerie
V
Uytterhoeven
VIB Center for Brain &Disease Research, Leuven, Belgium.|KU Leuven, Department of Human Genetics, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium.
Patrik
P
Verstreken
Structural Biology Research Center, VIB, Brussels, Belgium.|Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium.
Wim
W
Versées
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D019465:Craniofacial Abnormalities / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004058:Diffusion; D029721:Drosophila Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004827:Epilepsy / Q000235:genetics / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006228:Hand Deformities, Congenital / Q000235:genetics / Q000378:metabolism; D006319:Hearing Loss, Sensorineural / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008607:Intellectual Disability / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins; D008958:Models, Molecular; D009154:Mutation; D009264:Nails, Malformed / Q000235:genetics / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D000072417:Protein Domains; D013572:Synaptic Vesicles / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
D002352:Carrier Proteins; D029721:Drosophila Proteins; D020690:GTPase-Activating Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010716:Phosphatidylinositols; C552551:TBC1D24 protein, human; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases; C558398:sky protein, Drosophila; D020691:rab GTP-Binding Proteins
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