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1 Supporting Information Garcia-Marcos et al /pnas SI Methods: Bioinformatics Homology Modeling. The structure of the synthetic peptide KB- 752 bound to G i1 [PDB:1Y3A] was used as the template to generate the modeling project in Deep View/Swiss-PdbViewer v3.7 for GIV (aa ) in complex with G i3. The modeling project was submitted to the Swiss-Model Server (1), and model images were generated by MOLMOL. The modeling result was checked by Verify3D ( Verify 3D) (2) and WHATCHECK ( servers/html/index.html) (3) to identify possible errors. The parameters analyzed by these programs did not indicate significant errors in the modeled structure. residue repeated in the corresponding position at least twice among all of the different KB-752-related peptides. Since the sequence created using these criteria gave no other result than GIV in the search, we reduced the stringency by taking into account only the 7 residues corresponding to the core motif VTLQQFL of hgiv, which is highly conserved with the KB-752-related peptides. With these criteria, the degenerate sequence [MIVL]-[TS]-[WL]-[SYWQ]-[EDQ]-[FY]-[LI] was created and used in a MOTIF search of the Swiss-Prot database. The search returned 154 results, but none of them, other than GIV, was a known G -interacting protein. Although the GPCR T2R3 was found to have a related motif, this sequence was buried within the second membrane spanning region. Motif Search. hgiv s GEF motif (Accession #BAE44387, residues ) and the KB-752-related peptides described in ref. 4 were aligned using Clustal W. A degenerate sequence was created by having represented in each position of the sequence the corresponding residue present in GIV s GEF motif or any Secondary Structure Prediction. The secondary structure of the C terminus of GIV was predicted by using Jnet ( www-jpred/), PSIPRED ( bioinf.cs.ucl.ac.uk/psipred/psiform.html), or SSpro ( scratch.proteomics.ics. uci.edu/). 1. Schwede T, Kopp J, Guex N, Peitsch MC (2003) SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res 31: Luthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with threedimensional profiles. Nature 356: Hooft RW, Vriend G, Sander C, Abola EE (1996) Errors in protein structures. Nature 381: Johnston CA, et al. (2005) Structure of Galpha(i1) bound to a GDP-selective peptide provides insight into guanine nucleotide exchange. Structure (London)13: Kleuss C, Raw AS, Lee E, Sprang SR, Gilman AG (1994) Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc Natl Acad Sci USA 91: of7

2 Homo sapiens [BAE44387] 1623 GEFSLLHDHEAWSSSGSSPIQYLKRQTRSSPVLQHKISETLESR-HHKIKTGSPGSEVVTLQQFLEESNKLTSVQIKSSSQENLLDEVMKSLSVSSDFLGKDK-----PVSCGLARSVS Pan troglodyte [XP_ ] 1623 GEFSLLHDHEAWSSSGSSPIQYLKRQTRSSPVLQHKISETLESR-HHKIKTGSPGSEVVTLQQFLEESNKLTSIQIKSSSQENLLDEVMKSLSVSSDFLGKDK-----PVSCGLARSVS Mus musculus [Q5SNZ0.2] 1623 GDFSLLHDHETWSSSGSSPIQYLKRQTRSSPMLQHKISETIESRAHHKMKAGSPGSEVVTLQQFLEESNKLTSIQLKSSSQENLLDEVMKSLSVSSDFLGKDK-----PVSCTLARSVS Rattus norvegicus [XP_ ] 1597 GDFSLLHDHETWSSSGSSPVQYLKRQTRSSPMLQHKMSETVDSQAHHKMNAGSPGSEVVTLQQFLEESNKLTSIQLKSSSQENLLDEVMKSLSVSSDFLGKDK-----PVSCTLARSVS Ornithorhynchus anatinus [XP_ ] 1600 GEFSLLHDHDAWSSSSSSPIQYLKRNTRSSPGLQHKMPETLDGQGYHRIKTGSPGSEVVTLQQFLEESNKLTSVQIKSSSQENLLDEVMKSLSVSPDFMGREKTVKQSAVGCGISRSVS Gal lus gallus [X P_ ] 1599 GEFSMLHEHDAWSSSSSSPIQYLKGHTRSSPVLQQRTPETLDRC-GRQIKTDSPGSEVVTLQQFLEESNKSTSSEMKSGSEENLLDEVMRSLSESSELAGKEK-LRKASAGCGIVRSLS Tetraodon nigroviridis [CAF ] ] 638 GEFSLSLDQELWSSSGSSPVAQPSRSSRQSP-LQLR--KSLDPNASGSSPGQSRAGEVLSLQQFLDEGIDPAEVCQTTGT Danio rerio [AAI ] ] SESSGDVSVGVDAQLGAP DARRSAPSTGSDVVSLQQFLEEN-THTAEDPPSAPPSSRERVKARGILRSESRSGAGR Homo sapiens [BAE44387] GKTPGDFYDRRTTKP-EFLRPGPRKTEDTYFI-SSAGKPTPGTQGKIKLVKE---SSLS-RQSKDSNPYATLPRASSVISTAEGTTRRTSIHDFLTKDSRLPISVDSPPAAADSNTTA Pan troglodyte [XP_ ] GKTPGDFYDRRTTKP-EFLRPGPRKTEDTYFI-SSAGKPTPGTQGKIKLVKE---SSLS-RQSKDSNPYATLPRASSVISTAEGTTRRTSIHDFLTKDSRLPISVDSPPAAADSNTTA Mus musculus [Q5SNZ0.2] GKTPGDFYDRRTTKP-EFLRTGPQKTEDAYTI-SSAGKPTPSTQGKIKLVKE---TSVS-RQSKDSNPYATLPRASSVISTAEGTTRRTSIHDFLSKDSRLPVSVDSSPPTAGSSSTT R attus norvegicus [X P_ /] GKAPGDFYDRRTAKP-EFLRTGPQKTEDAYSI-SSAGKPTPSTQGKIKLVKE---TSVL-RQSKDSNPYATLPRASSVISTAEGTTRRTSIHDFLSKDSRLPVSVEPAPPPAGGSSTA Ornithorhynchus anatinus [XP_ ] VRCTTDFSDGKPTKPEQFVLPNPRKTEDSYFS-SSSGK--SGTQTKVKLVKE---TSLSQRQSKDHNPYATLPRASSVISTAEGTTRRTSIHDFLSKDSRQPVSIDPSPSTTDSTFSS Gal lus gallus [XP_ ] VKNPVDFSEGRSIKPEQLVRPSLRRTEDAYFT-SSPIKFTSGTQGKAKSVKEMMQTSVSQRQSRDCNPYATLPRASSVISTAEGTTRRTSIHDFLSKDIRQPASGDPATSTADRSVPA Tetraodon nigroviridis [CAF ] PHLRKAESTRVRGSVPIRPSLSSQGKATSVSE RLDSAS---STLPRASSVISTAEGSTRRTSIHDMLSKDSRQPVSAGPRPQPTPSEYHS Danio rerio [AAI ] PSLRKTESTRARGCA-PPRAGSATQRAASVSALD SSGLPRASSVISTAEGSVRRTSIHDLLSRDSRQPVLVDPPALRSGSSNVE Homo sapiens [BAE44387] ASNVDKVQESRNSKSRSREQQSS 1870 Pan troglodyte [XP_ ] AS Mus musculus [Q5SNZ0.2] ASNVNKVQESRNSKSRSREQQSS 1871 Rattus norvegicus [XP_ ] ASNVNKVQESRNSKSRSREQQSS 1845 Ornithorhynchus anatinus [XP_ ] TSNVEAIQESRNSKSRSREQQSS 1852 Gallus gallus [XP_ ] TSN Tetraodon nigroviridis [CAF ] NSSALKV Danio rerio [AAI ] NRKSKSR Fig. S1. Sequence alignment of GIV homologues. The sequence corresponding to the C-terminal domain of human GIV (BAE44387, aa ) was used to identify homologues by BLAST search. The identified homologues with higher identity scores were aligned using CLUSTAL W. Conserved residues are shaded in black; similar residues in gray. Accession number for each species is indicated in brackets. The result of the alignment showed two stretches of residues (indicated with red boxes) conserved across species from fish to humans. 2of7

3 Fig. S2. Secondary structure prediction and homology model of GIV s GEF motif bound GDP G i3. (A) The predicted secondary structure of GIV s GEF motif is compatible with the secondary structure of the KB-752 peptide. The secondary structure of GIV s GEF motif was predicted by Jnet, PSIPRED, or SSpro. The consensus among the predictions by the different programs predicts a partially -helical conformation analogous to that observed in the KB-752 peptide in the complex with G i1 (upper red line). -helix, -strand, - unstructured. (B) The structure of the synthetic peptide KB-752 bound to G i1 [PDB:1Y3A] was used to generate the template for modeling that was submitted to the Swiss-Model Server. G i3 all-helical domain is shown in yellow, ras-like domain in blue and switch regions in green. The GEF motif identified in GIV is shown in red. The arrow denotes the proposed escape route for GDP (4) (C) Analysis of the modeling result by Verify3D. Verify3D was used to evaluate the compatibility between the spatial localization of the aminoacids in the 3-dimensional model (GIV G i3) and its own primary sequence. The 3-dimensional structure of the template (KB-752 G i1 crystal structure) was also analyzed for comparison. The S score of the model was high and similar to the template indicating the modeled structure has a good compatibility with the primary sequence. 3D-1D scores were always above 0.1 values, indicating that no problematic region is found in the model. (D) The correctness of the model was evaluated by WHATCHECK and the root mean square deviation (RMSD) (residues of G i3 and of GIV for the Interface backbone ) calculated by Deep View/Swiss-PdbViewer v3.7. The parameters analyzed did not indicate significant errors in the modeled structure, indicating a valid model with high reliability. 3of7

4 Fig. S3. GIV-CT and KB-752 peptide compete for binding to G i3. (A) A fixed amount of His-GIV-CT (2.4 g) and the indicated amounts of the KB-752 peptide (0 30 M) were incubated with 7 g of GST-G i3 preloaded with GDP immobilized on glutathione beads. Bound proteins were quantified by His immunoblotting (IB) and equal loading of GST-G i3 protein confirmed by protein staining with Ponceau S. (B) His-GIV-CT binding to GST-G i3 was expressed as percent of the amount bound in the absence of KB-752. Results from 3 independent experiments were averaged and fitted to a sigmoidal dose curve using Prism software. 4of7

5 Fig. S4. Endogenous GIV and recombinant His-GIV-CT show similar binding to different G i-subunits. COS-7 cell lysates (A) or purified His-GIV-CT (B) were incubated with approximately 15 g GST-G i1, GST-G i2, GST-G i3, or GST previously loaded with GDP or GDP AlF 4 and immobilized on glutathione beads. Bound proteins were analyzed by immunoblotting (IB). Equal loading of GST proteins was confirmed by protein staining with Ponceau S. 5of7

6 Fig. S5. Gallein inhibits GIV-mediated enhancement of Akt phosphorylation in a dose-dependent manner. COS-7 cells were transfected with GIV plasmid or vector control. Forty-eight hours after transfection, cells were incubated with the indicated concentrations of gallein ( M) or DMSO for 30 min in the presence of 10% serum. Cells were lysed and analyzed for GIV, actin, phospho-akt (pakt), and total Akt (takt) by immunoblotting (IB). The ratios of pakt to takt were determined for the different gallein concentrations and fitted to a sigmoidal dose curve using Prism software to estimate the IC 50 (approximately 3 M). 6of7

7 Fig. S6. Quality control of the His-G i3 protein used in this work. (A) His-G i3 was purified as described in Materials and Methods. Twenty-five micrograms of protein were resolved by 10% SDS/PAGE gel and stained with Coomassie Blue. The purity of the His-G i3 preparation was estimated to be 95%. (B) The correct folding and stability of the protein were assessed by the trypsin protection assay as described in (5). Briefly, His-G i3 (1 mg/ml) was incubated for 120 min at 30 C in the absence of nucleotide or presence of GDP (30 M) or GDP AlF 4 (30 M GDP, 30 M AlCl 3, 10 mm NaF) as indicated. After this incubation, trypsin was added to the tubes at a final concentration of 25 g/ml and samples were incubated for an additional period of 10 min at 30 C. Reactions were stopped by addition of SDS/PAGE sample buffer to the tubes and boiling. Proteins were resolved by SDS/PAGE and stained with Coomassie blue as in (A). Asterisk (*) denotes the trypsinization-resistant form of active, GDP AlF 4 -loaded His-G i3, indicating that the protein is properly folded and can undergo a conformational change upon activation (5). 7of7