The Skap-hom Dimerization and PH Domains Comprise

Transcription

1 Molecular Cell, Volume 32 Supplemental Data The Skap-hom Dimerization and PH Domains Comprise a 3 -Phosphoinositide-Gated Molecular Switch Kenneth D. Swanson, Yong Tang, Derek F. Ceccarelli, Florence Poy, Jan P. Sliwa, Benjamin G. Neel, and Michael J. Eck Table S1. Dynamic Light Scattering (DLS) analysis of selected Skap-hom and Skap55 protein fragments. Protein Names (residues) Calculated MW (Da) DLS MW* (Da) Skap-hom DM-PH (14-222) Skap55 DM-PH (7-213) Skap-hom PH ( ) Skap55 PH (98-213) 23,993 24,008 14,245 13,625 70,600 60,900 16,300 16,400 *Note that DLS does not provide a direct measure of molecular mass. The estimated molecular weights are based on globular protein standards.

2 Figure S1. Superdex 75 analytical gel filtration chromatography (Left Panel) and Coomassiestained SDS-PAGE analysis (Right Panel) of the DM-PH and PH fragments of Skap-hom and Skap55 proteins. The DM-PH constructs of both Skap-hom (peak 1) and Skap55 (peak 2) elute from the gel-filtration column at approximately the volume expected for dimeric species, while the isolated PH domains elute as expected for monomers (peaks 3 and 4, for Skap-hom and Skap55, respectively). It is not clear why the Skap-hom proteins elute somewhat earlier than their Skap55 counterparts.

3 Figure S2. An alternate view of the DM/PH domain interface. The PH domain is shown in red and the DM domain in blue as in Figure 2A. Salt-bridges are indicated by dashed lines (red, intra-domain; black, inter-domain).

4 Figure S3. Functional analysis of full length GFP-tagged DM* mutants. (A) Flag-tagged WT Skap-hom was expressed in 293T cells with WT Skap-hom-GFP (lane 1), WT Skap-hom plus Skap-hom-Flag, (lane 2) pmxpuro plus Skap-hom-Flag (lane 3), DM* mutant Skap-hom-GFP plus pmscv (lane 4), and DM* mutant Skap-hom-GFP plus Skap-hom-Flag (lane 5). Lysates from these cells were subjected to immunoprecipitation using M2 flag agarose and immunoblotted with anti-skap-hom antibodies. (B) Constructs encoding WT Skap-hom-GFP or DM* mutant Skap-hom-GFP were co-transfected into 293T cells with HA-tagged Adap or with empty vectors, as indicated. Lysates from the transfected cells were subjected to anti-ha immunoprecipitation and analyzed by immunoblotted using anti-ha (top panel) or anti-skaphom (lower panel).

5 Figure S4. Immunostaining of WT and Skap-hom -/- BMM with anti-skap-hom antibodies. WT (A, C and E) and Skap-hom -/- (B, D and F) BMM were stained with anti-skap-hom antibodies (A and B) and rhodamine phalloidin (C and D). Merged images are shown in panels E and F.

6 Figure S5. Inhibition of PI 3-kinase results in re-localization of Skap-hom away from actin ruffles. BMM expressing GFP-Skap-hom were treated with DMSO for 30 minutes (A - C) or 15μm LY for 10 (D-F) or 30 (G-I) minutes. F-actin was visualized by rhodamine phalloidin staining (A, D, G) and the localization of the expressed GFP-Skap-hom was detected by green fluorescence (B, E, H). Merged images show the differences in F-actin and Skap-hom localization (C, F, I).

7 Figure S6. Stills from movies of live Skap-hom -/- BMM expressing GFP-fused WT or mutant Skap-hom proteins. (A) GFP control, (B-E) WT Skap-hom-GFP, (F-J) DM* Skap-hom-GFP, (K- O) R140M-Skap-hom-GFP, (P) D129K Skap-hom GFP, (Q) D129K:R140M Skap-hom-GFP, (R) K56A-Skap-hom-GFP, (S) K56A:R140M-Skap-hom-GFP, and (T) ΔPH-Skap-hom-GFP. Images presented are representative; for each variant between 14 and 82 fields were imaged.

8 Figure S7. Model for the 3 phosphoinositide-gating of Skap-hom. (A) In the absence of binding to 3 phosphoinositides, the Skap-hom PH domains are docked against the dimerization domain in a manner that perturbs their phosphoinositide binding pockets. (B) A free state that is competent to bind 3 phosphoinositides may exist in equilibrium with the docked state. (C) Binding of 3 -phosphoinositides is expected to stabilize the free state, and to thereby expose a putative protein-protein interaction site that results in localization of Skap-hom to actin-rich ruffles.

9 Supplemental Experimental Procedures Preparation and characterization of selected Skap-hom and Skap55 protein fragments Skap-hom fragments containing the N-terminal predicted coiled-coil, the first linker (L1), and the PH domain (residues , termed DM-PH), or the PH domain alone (residues ), or the equivalent fragments of Skap55 (residues and , respectively) were produced in bacteria and purified to apparent homogeneity and analyzed by gel filtration with a Superdex 75 analytical column (GE Healthcare). The isolated PH domains of Skap-hom and Skap55 eluted at 13.2 ml and 13.8 ml, respectively, consistent with a monomeric species of 14 KDa molecular weight. In contrast, the longer fragments of each protein eluted much earlier than expected for proteins with their larger monomeric molecular mass, suggesting oligomerization (standard proteins elution volumes were indicated for reference). Single clear elution profiles of both proteins during gel filtration clearly indicate that there is only one single oligomeric state of both longer protein fragments. Dynamic Light scattering (DLS) experiments were carried out with a Protein Solutions DLS instrument. Structure Determination of the IU5F Skap-hom PH domain We initially determined the structure of a Skap-hom protein that contained 11 vector-derived residues (RASVGSPGIPA) appended to the N-terminus of the protein. The vector-derived residues in this structure artifactually occluded the phophoinositide-binding pocket; thus, this structure is not presented in the text. This structure was, however, used as a molecular replacement model to phase the structures presented here; therefore, we briefly describe its elucidation below. We refer to this structure by its PDB ID code, 1U5F.

10 Isomorphous heavy-atom derivative crystals of 1U5F were prepared by soaking the native crystals for 2 hours in a mother liquor containing 2.0M ammonium sulfate, 100 mm sodium citrate ph 5.5, 20% glycerol, and 2 mm methyl-mercury nitrate. The structure of 1U5F was solved by using the Single Isomorphous Replacement with Anomalous Scattering (SIRAS) method. The position of the heavy atom in the methyl-mercury nitrate derivative was determined with both the isomorphous difference and the anomalous difference Patterson maps calculated with Å data using the CCP4 suite (CCP4, 1994). The heavy-atom parameters were refined and the SIRAS phases were calculated to 2.3 Å resolution with the program MLPHARE (CCP4, 1994). The initial phases were improved by solvent flattening and histogram matching with the program DM in the CCP4 suite (CCP4, 1994). Solvent-flattened maps at various resolutions were generated and used in the model building. An ab initio model was built with the program O (Jones et al., 1991). The 1U5F mainchain backbone was traced before a complete model was generated using the protein sequence. The model was refined using a combination of molecular dynamics, positional and individual B factors refinements that gradually extended to 1.9 Å resolution. All refinements were performed with the program CNS (Brunger et al., 1998) using the stereochemical parameters of Engh and Huber (Engh and Huber, 1991). Difference or omit electron density maps were calculated and inspected after each refinement and the model was adjusted manually wherever necessary. Waters were added and refined to verify accuracy at the final stage of the refinement. The final model was refined to a resolution of 1.9 Ǻ with a crystallographic R factor of 22.6% (R free =24.6%) and r.m.s. deviations of Ǻ (bond-length) and degree (bond angles), respectively. More statistics are available in the Protein Data Bank deposition ID 1U5F.

11 Supplemental References Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., et al. (1998). Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr D Biol Crystallogr 54, CCP4 (1994). The CCP4 suite: programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, Engh, R. A., and Huber, R. (1991). Accurate bond and angle parameters for X-ray protein structure refinement. Acta Cryst A47, Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991). Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47 ( Pt 2),