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

Nature Structural & Molecular Biology volume 23, pages 965–973 (2016)Cite this article

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Abstract

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)P2 concentrations 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.

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Figure 1: Crystal structure of the unconventional TBC domain of Sky (Sky1–353).
Figure 2: Disease-associated mutations in TBC1D24-Sky.
Figure 3: The cationic pocket of the TBC domain of Sky binds phosphoinositides.
Figure 4: Mutations in the cationic pocket increase Sky protein diffusion in synaptic terminals.
Figure 5: The cationic pocket in Sky is required for presynaptic function.
Figure 6: Mutation of the cationic pocket of Sky induces susceptibility to seizures, loss of coordination and hyperactivity in adult flies.
Figure 7: Partial loss of synj rescues Sky with cationic-pocket mutations.

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Acknowledgements

We thank the staff at the beamlines Proxima 2 of Soleil (France) and ID23-1 of ESRF (France) for assistance during data collection. We thank E. Lauwers and J. McInnes for advice on lipid biology and members of the Versées and Verstreken laboratories for comments. This work was supported by the Fonds voor Wetenschappelijk Onderzoek (J.P., V.U., P.V. and W.V.), Strategic Research Program Financing from the VUB (W.V.) and KU Leuven (P.V.), the Hercules foundation (W.V. and P.V.), BioStruct-X from the European Community's Seventh Framework Programme (W.V.), the ERC (P.V.), a Methusalem grant from the Flemish government (P.V.), the Belgian Science Policy (P.V.), Agentschap voor Innovatie door Wetenschap en Technologie (I.M.), the Belgian American Educational Foundation (K.L.) and VIB (P.V.).

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  1. Baptiste Fischer, Kevin Lüthy and Jone Paesmans: These authors contributed equally to this work.

Authors and Affiliations

  1. Structural Biology Research Center, VIB, Brussels, Belgium.,

    Baptiste Fischer, Jone Paesmans, Charlotte De Koninck & Wim Versées

  2. Structural Biology Brussels, Vrije Universiteit Brussel, Brussels, Belgium

    Baptiste Fischer, Jone Paesmans, Charlotte De Koninck & Wim Versées

  3. VIB Center for Brain & Disease Research, Leuven, Belgium

    Kevin Lüthy, Ine Maes, Jef Swerts, Sabine Kuenen, Valerie Uytterhoeven & Patrik Verstreken

  4. Department of Human Genetics, KU Leuven, Leuven Institute for Neurodegenerative Disease (LIND), Leuven, Belgium

    Kevin Lüthy, Ine Maes, Jef Swerts, Sabine Kuenen, Valerie Uytterhoeven & Patrik Verstreken

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Contributions

B.F. and J.P. solved the structures, produced and purified proteins, performed biochemical experiments, analyzed data and wrote the manuscript; K.L. performed biochemical and in vivo experiments, analyzed data and wrote the manuscript; C.D.K. assisted with biochemistry; I.M., S.K. and V.U. performed in vivo experiments; J.S. performed transmission electron microscopy; P.V. and W.V. conceived and supervised the work, contributed to data interpretation and wrote the manuscript. All authors reviewed the manuscript.

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Correspondence to Patrik Verstreken or Wim Versées.

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Integrated supplementary information

Supplementary Figure 1 Different crystal forms obtained for Sky1–353.

(a) Crystal form 1 obtained in the presence of 20% PEG 3350 and 0.2 M ammonium citrate tribasic pH 7.0.

(b) Crystal form 1 of the selenomethionine-labelled Sky1-353 obtained in the presence of 20% PEG 3350 and 0.2 M ammonium citrate tribasic pH 7.0

(c) Crystal form 2 obtained in the presence of 25% PEG 1500 and 0.1 M succinate/phosphate/glycine pH 7.0. This crystal form was used to obtain the Sky1-353-IP3 crystal structure after soaking with 5 mM IP3.

Supplementary Figure 2 Sequence alignment of Sky1–353 with the TBC domain of human TBC1D24 and representative RabGAP proteins for which the crystal structure has been reported.

The residues corresponding to the arginine and glutamine fingers in conventional TBC Rab-GAP proteins are highlighted in green. The residues corresponding to the cationic pocket residues in Sky are highlighted in blue. Residues corresponding to patient mutations are red, with those located in the cationic pocket additionally indicated by a red star. The α-helices determined from the Sky1-353 structure are indicated above the alignment, with the numbering corresponding to the numbering used in the Gyp1 structure. The sequences used are human TBC1D24 (UniProt: Q9ULP9), Gyp1 from Saccharomyces cerevisiae (UniProt: Q08484), human TBC1D1 (UniProt: Q86TI0), human TBC1D4 (Uniprot: O60343), human TBC1D7 (UniProt: Q9P0N9), human TBC1D11 (UniProt: Q9Y3P9), human TBC1D14 (UniProt: Q9P2M4), human TBC1D18 (UniProt: Q5R372), human TBC1D20 (UniProt: Q96BZ9), human TBC1D22A (UniProt: Q8WUA7), human TBC1D22B (UniProt: Q9NU19), CrfRabGAP from Chlamydomonas reinhardtii (UniProt: A8JCA4).

Supplementary Figure 3 Surface electrostatics of Sky1–353 in comparison to other TBC domains.

The electrostatic potential mapped on the solvent accessible surface of Sky1-353 in the first panel shows the cationic pocket located on the opposite side of the GTPase binding region. The electrostatic potential surfaces of the 11 other TBC domains deposited in the PDB are shown in exactly the same orientation. None of these proteins seems to harbor a well-defined cationic pocket similar to Sky1-353. The structures of the TBC domains shown in this figure are: Gyp1 (pdb 1FKM; Rak, A. et al., EMBO J. 19, 5105–13, 2000), TBC1D1 (pdb 3QYE; Park, S.-Y. et al., J. Biol. Chem. 286, 18130–8, 2011), TBC1D4 (pdb 3QYB; Park, S.-Y. et al., J. Biol. Chem. 286, 18130–8, 2011), TBC1D7 (pdb 3QWL; unpublished), TBC1D11 (pdb 4NC6; unpublished), TBC1D14 (pdb 2QQ8; unpublished), TBC1D18 (pdb 3HZJ; unpublished), TBC1D20 (pdb 4HL4; Gavriljuk, K. et al., Proc. Natl. Acad. Sci. U.S.A. 109, 21348–53, 2012), TBC1D22A (pdb 2QFZ; unpublished), TBC1D22B (pdb 3DZX; unpublished), CrfRabGAP (pdb 4P17; Bhogaraju, S. & Lorentzen, E. Proteins 82, 2282–7, 2014).

Supplementary Figure 4 Influence of PI(4,5)P2 and salt concentrations on the binding of Sky1–353 to liposomes.

(a) Western blots of liposome flotation assays using wild type Sky1-353 and liposomes (PC:PS) enriched with different concentrations of PI(4,5)P2 (0%, 0.5%, 2% and 5%). n = 2 independent experiments.

(b) Western blots of liposome flotation assays using wild type Sky1-353 and liposomes (PC:PS) enriched with 2% of PI(4,5)P2 in the presence of three different NaCl concentrations (30 mM, 150 mM and 500 mM). n = 2 independent experiments.

Original blots can be found in Supplementary Data Set 1.

Supplementary Figure 5 Comparison of the phosphoinositide-binding pocket of Sky1–353 with other typical phosphoinositide-binding domains.

Structures of representatives of well-established phosphoinositide-binding domains (ENTH, PX, FYVE and PH) in complex with a phosphoinositide head group are shown in electrostatic surface representation and are compared to the Sky1-353 structure in complex with IP3. The boxes show a close-up of the phosphoinositide binding pocket in cartoon representation with the phosphoinositide head group and the interacting residues shown as sticks. The structures that are shown are: the TBC domain of Sky bound to IP3 (this study), the ENTH domain of epsin bound to IP3 (pdb 1H0A; Ford, M. G. J. et al., Nature 419, 361–366, 2002), the PX domain of P40phox bound to dibutanoyl IP2 (pdb 1H6H; Bravo, J. et al., Mol. Cell 8, 829–39, 2001), the FYVE domain of EEA1 bound to IP2 (pdb 1JOC; Dumas, J. J. et al., Mol. Cell 8, 947–58, 2001), the PH domain of PLC-δ1 bound to IP3 (pdb 1MAI; Ferguson, K. M. et al., Cell 83, 1037–1046, 1995) and the PH domain of spectrin bound to IP3 (pdb 1BTN; Hyvönen, M. et al., EMBO J. 14, 4676–85, 1995).

Supplementary Figure 6 Wild-type and mutant Skywalker-GFP traffic to and are present at synaptic terminals

(a) Confocal images of the ventral nerve cord in third instar Drosophila larvae expressing wild type GFP-Sky (SkyWT) or mutant GFP-Sky (SkyR79C, SkyR281C or Sky3Glu). Scale bar for all top panels: 20 μm.

(b) Confocal images of larval neuromuscular junction endplates of animals with genotypes as in (a). (n = 6 animals), scale bar for all panels in (b): 20 μm. GFP intensities between the GFP-Sky mutants is not significantly different.

(c) Quantification of third-instar NMJ GFP intensity at the membrane in single confocal sections. Mean Fluorescence Intensity between the genotypes GFP-SkyWT, GFP-SkyR79C and GFP-Sky3Glu is similar. Error bars: mean ± s.e.m. (n = 6) P = 0.446, ns: not significant by ANOVA, Dunnett’s.

(d) Quantification of Western blot band intensity of GFP-Sky and GFP-Syt in protein isolations from adult fly heads. Intensities are normalized to Syntaxin control levels. GFP-Sky WT and mutant levels are similar. Error bars: mean ± s.e.m. (n = 3) P = 0.681, ns: not significant by ANOVA, Dunnett’s. The levels of Synaptotagmin-GFP (Syt) are shown as a control.

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Supplementary Figures 1–6 (PDF 1336 kb)

Supplementary Data Set 1

Uncropped blots for Figure 3 and Supplementary Figure 4. (PDF 3079 kb)

Seizure asssay of Drosophila carrying mutations in the cationic pocket of Sky.

Movie of behavior of adult sky1/2 Drosophila males (yw eyFLP/Y; FRT40A sky1:FRT40A sky2) expressing GFP-SkyWT, GFP-SkyR79C or GFP-Sky3Glu using nSybGal4, following 10 seconds of vortex stimulation. (MP4 1269 kb)

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Fischer, B., Lüthy, K., Paesmans, J. et al. Skywalker-TBC1D24 has a lipid-binding pocket mutated in epilepsy and required for synaptic function. Nat Struct Mol Biol 23, 965–973 (2016). https://doi.org/10.1038/nsmb.3297

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