Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1. Different crystal forms obtained for Sky

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Randell Harper
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Supplementary Figure 1 Different crystal forms obtained for Sky 1 353. (a) Crystal form 1 obtained in the presence of 20%

1 Supplementary Figure 1 Different crystal forms obtained for Sky (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 Sky 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 Sky IP 3 crystal structure after soaking with 5 mm IP 3.

3 Supplementary Figure 2 Sequence alignment of Sky 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 Sky 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 Sky 1 353 in comparison to other TBC domains.

4 Supplementary Figure 3 Surface electrostatics of Sky in comparison to other TBC domains. The electrostatic potential mapped on the solvent accessible surface of Sky 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 Sky The structures of the TBC domains shown in this figure are: Gyp1 (pdb 1FKM; Rak, A. et al., EMBO J. 19, , 2000), TBC1D1 (pdb 3QYE; Park, S.-Y. et al., J. Biol. Chem. 286, , 2011), TBC1D4 (pdb 3QYB; Park, S.-Y. et al., J. Biol. Chem. 286, , 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, , 2012), TBC1D22A (pdb 2QFZ; unpublished), TBC1D22B (pdb 3DZX; unpublished), CrfRabGAP (pdb 4P17; Bhogaraju, S. & Lorentzen, E. Proteins 82, , 2014).

Supplementary Figure 4 Influence of PI(4,5)P 2 and salt concentrations on the binding of Sky 1 353 to liposomes.

5 Supplementary Figure 4 Influence of PI(4,5)P 2 and salt concentrations on the binding of Sky to liposomes. (a) Western blots of liposome flotation assays using wild type Sky and liposomes (PC:PS) enriched with different concentrations of PI(4,5)P 2 (0%, 0.5%, 2% and 5%). n = 2 independent experiments. (b) Western blots of liposome flotation assays using wild type Sky and liposomes (PC:PS) enriched with 2% of PI(4,5)P 2 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 Sky 1 353 with other typical phosphoinositide-binding domains.

6 Supplementary Figure 5 Comparison of the phosphoinositide-binding pocket of Sky 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 Sky structure in complex with IP 3. 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 IP 3 (this study), the ENTH domain of epsin bound to IP 3 (pdb 1H0A; Ford, M. G. J. et al., Nature 419, , 2002), the PX domain of P40 phox bound to dibutanoyl IP 2 (pdb 1H6H; Bravo, J. et al., Mol. Cell 8, , 2001), the FYVE domain of EEA1 bound to IP 2 (pdb 1JOC; Dumas, J. J. et al., Mol. Cell 8, , 2001), the PH domain of PLC-δ1 bound to IP 3 (pdb 1MAI; Ferguson, K. M. et al., Cell 83, , 1995) and the PH domain of spectrin bound to IP 3 (pdb 1BTN; Hyvönen, M. et al., EMBO J. 14, , 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

7 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 (Sky WT ) or mutant GFP- Sky (Sky R79C, Sky R281C or Sky 3Glu ). Scale bar for all top panels: 20 m. (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 m. 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-Sky WT, GFP-Sky R79C and GFP-Sky 3Glu is similar. Error bars: mean ± s.e.m. (n = 6) P = 0.446, ns: not significant by

8 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.

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1 Supplementary Figure 1 Crystallization. a, Crystallization constructs of the ET B receptor are shown, with all of the modifications to the human wild-type the ET B receptor indicated. Residues interacting