Supplemental Material Liu et al.

Transcription

1 Supplemental Material Liu et al. Supplemental Methods Protein expression, purification Recombinant etud11 (a.a ) and Tud7-11 (a.a ) proteins (Tud7-11) were expressed in the Rosetta strain of E. coli at 18 C using the pet28-smt3 vector. The N-terminal 6xHis-SUMO tagged proteins were first purified on a Ni-chelating column, and the tag was subsequently removed by digestion with SUMO protease. The tagless etud11 was concentrated to ~20 mg/ml for crystallization, and Tud7-11 was further purified on a Superdex-200 column. Selenomethionine (SeMet) substituted etud11 was expressed in the BL21(DE3) strain of E. coli using a defined medium supplemented with 60 mg/l of SeMet. The N2432A point mutation was generated with the MutanBest Kit (TAKARA) according to the manufacturer s protocol. Both the SeMet and the N2432 mutant proteins were purified following the same procedure as that for the wild-type protein. Surface plasmon resonance (SPR) binding assay Binding experiments were performed using BIAcore 3000 (GE Healthcare). Biotin-labeled Aub peptides were coupled to SA biosensor chips, and Tud7-11 (5 µm) or etud11 (40 µm) was passed through flow cells of peptide-coupled chips in a running buffer (20 mm Tris-HCl ph 8.0, 200 mm NaCl, 0.005% Tween 20) at a flow rate of 30 µl/min. The association and disassociation time were 60 s and 150 s, respectively. The biosensor chips were regenerated with 30 µl of 2M NaCl solution or 15 µl of 20 mm NaOH between injections. Figures were prepared using the BIAevaluation software. The binding curves of Tud7-11 to Aub peptides were generated using two SA-chips. The first flow cells of both chips were blank control. Btn-Aub, Btn-Aub[R11(me2s)] and Btn-Aub[R13(me2s)] peptides were immobilized on three flow cells of one chip at levels of 427, 440 and 420 RUs, respectively. Btn-Aub, Btn-Aub[R13(me2s)] and Btn-Aub[R15(me2s)] peptides were immobilized on a second chip at levels of 408, 410 and 418 RUs, respectively. The response data of Btn-Aub[R13(me2s)] from the two chips were used for normalization of responses across chips. Site-directed mutagenesis Mutant Tud(D7-11) transgenes were generated by PCR. Point mutations, replacing pairs of aromatic amino acids in each Tudor domain were made either single (D7*, D8*, etc) or combined (D7-11*) using the QuikChange Multi site-directed mutagenesis kit (Stratagene): D7*: Y1680A, Y1697A; D8*: Y1857A, Y1877A; D9*: Y2041A, Y2061A; D10*: Y2227A, Y2248A; D11*: Y2410A, F2430A. Sequence of oligonucleotides used for mutagenesis will be provided on request. Quantification of localized Aub and HA signals The intensity of localized Tud-HA or Aub staining in egg chambers was measured using 1

2 ImageJ. Seven reads of localized HA or Aub were collected per egg chamber. Eleven to sixteen egg chambers were analyzed for each genotype. Fas III staining of polar cells was used as internal standard for levels of posterior localization. An arbitrary intensity scale, set as 100%, was used for the intensity of the localized signal of wild-type Tudor domain 7-11 transgene. Quantification of Tud-HA expression Ovaries were dissected in cold 1X PBS and flash frozen in liquid nitrogen. Once thawed, the ovaries were homogenized with lysis buffer (50 mm Tris-base at ph 8.0, 150 mm NaCl, EDTA-free protease inhibitor cocktail tablet, Roche) to extract the supernatant. Western blotting was done as described (Bardsley et al. 1993). Rat anti-ha [clone 3F10] (Roche Applied Science) and rabbit anti-kinesin heavy chain (KHC) (Cytoskeleton, Inc.) were used at 1:1000 and 1: IRDye 800CW goat anti-rat and donkey anti-rabbit (LI-COR Biosciences) secondary were used at 1: Signals were detected with LI-COR imaging systems. For quantification, Tud-HA signals were normalized to that of KHC. Electron microscopy Stage 1 embryos derived from wild-type and tud 1 /Df(2R)Pu rp133, Tud(D7-11) females were fixed, sectioned and contrasted for electron microscopy as described (Arkov et al. 2006). 2

3 Table S1: Crystallographic Analysis Statistics. Values in parentheses are those for the highest-resolution shell of reflections. Diffraction Data Data sets (Spacegroup) Complex R13me2s (P ) a Complex R15me2s (P ) b Apo Native Apo SeMet (C2) (C2) c Peak Inflection Remote Wavelength (Å) Resolution (Å) ( ) ( ) ( ) ( ) ( ) ( ) Total/Uniq. Obs / / / / / /27813 Completeness (%) 96.4 (94.6) 99.9 (100) 96.2 (80.3) 99.1 (99.9) 99.7 (99.8) 99.0 (93.8) R merge d (%) 9.8 (40.1) 6.2 (32.6) 5.6 (23.6) 5.1 (7.7) 4.1 (9.6) 4.0 (15.5) <I/σI> 14.7 (2.5) 22.4 (4.0) 21.2 (2.4) 30.5 (13.7) 30.4 (8.1) 31.7 (4.7) Redundancy 4.5 (4.5) 5.4 (5.5) 3.3 (2.5) 3.4 (3.4) 3.5 (3.5) 3.5 (3.2) Refinement Resolution (Å) No. of Obs R-work e (%) R-free e (%) R.m.s. deviations Bond length (Å) Bond angles (º) B factor (Å 2 ) Protein Peptide Water Numbers of atoms Protein Peptide Water Ramachandran plot stats Most favor. region 83.9% 83.2% 91.8% Disallowed region none none none a Cell dimensions: a=b=48.0 Å, c=152.0 Å. b Cell dimensions: a=b=48.8 Å, c=152.2 Å. c Cell dimensions: a=104.2 Å, b=47.3 Å, c=85.4 Å, and β= d R merge = Σ I - <I> /Σ<I>, where I and <I> are the averaged intensity of multiple measurements of the same reflection. The summation is over all the observed reflections. e R-factor = Σ F O - F C /Σ F O, where F O denotes the observed structure factor amplitude and F C denotes the structure factor calculated from the model. R-work was calculated using the diffraction data used throughout the refinement, and R-free was calculated using the 10% (native) or 5% (complexes) of the data set aside during refinement. 3

4 Liu et al. Figure S1. Tud7-11 transgene and mutant derivatives in germ cell formation and Aub localization. This is an expanded version of Fig.2 in the main text, including results for Tudor protein localization of the Tud7-11 wild-type and mutant transgenes. Please note, panels (A -F ) correspond to panels (A-F) in Fig. 2 and panels J-K correspond to G-H; M is I and O-Q are J-L in Fig. 2. They are included here for side-by-side comparison. (A)-(G) Expression of Tudor transgenes in stage 10 eggchambers stained with HA antibody to detect HA-tagged Tud7-11 (green) and DAPI for DNA (blue). (A) wild-type and (B)-(G), mutant Tud7-11 transgenes. In (B)-(F), the second and fourth aromatic residues in a single Tudor domain were changed to alanines (see Fig. 4 and Methods for aa mutated) and in G each domain is mutated. HA (green) and DAPI staining was used to detect transgene products and label DNA, respectively. Although all transgenes were inserted into the same genomic region, protein expression level varies (Fig. S2). (H) Quantitation of localized Tudor transgenic protein. Fas III staining in the polar cells (stainings not shown) was used as an internal standard for levels of posterior localization. (N=11~13 for each genotype). (A )-(G ) Stage 4 embryos stained with anti-vasa (green) to detect germ cell formation. (J)-(R) Aub localization in the wild-type (J), tudor mutant (K), tudor mutant with a full-length tudor transgene (L), tudor mutant with a wild-type (M) and single Tud domain mutant (N-R) 4

5 tud7-11 transgene. Stage 10 oocytes stained with Aub (green) and FasIII antibodies. Note that Tud7-11 is sufficient for localization of Aub to the posterior pole of the oocyte, but only the full-length tudor transgene restores Aub localization to the nuage of nurse cells. (S) Quantitation of Aub posterior pole localization in mutants and transgenic egg chambers. For quantitation, Fas III staining in polar cells (arrow in J) was used as an internal standard for levels of posterior. (N= 12~16 for each genotype). Note that Aub localization and stability (Supplemental Fig. 1) are affected similarly when the aromatic cage in Tudor domain D8 or D10 is mutated but only D10 affects germ cell formation, suggesting that Aub localization is not sufficient for germ cell formation. All egg chambers and embryos are oriented anterior to left. Figure S2. Protein expression levels and polar granule formation in transgeneic flies. (A) Protein Expression levels of Tudor-HA transgenes detected using Western blotting of ovary extracts probed with HA antibody. For normalization, blot was probed with anti-heavy Chain Kinesin antibody, and Tud/KHC values are given with the D7-11 wild-type transgene expression level set arbitrarily at 1. (B) Electron micrographs of germ plasm in wild type and tudor deficient embryos carrying D7-11 transgenes. In tud1/df; D7-D11(wt) polar granules were much reduced in size and were detected less frequently than that in the wild-type, suggesting that fully-formed polar granules are not required for localization of germ plasm RNA and proteins or germ cell formation 24. Polar granules are marked by arrows, and the letter m indicates mitochondria. 5

6 Figure S3. Binding of the etud11 protein to Aub peptides measured by SPR. The curves corresponding to Aub peptides with Arg15(me2s), Arg13(me2s) or no modifications are shown in magenta, red and blue, respectively. The immobilize amounts of Btn-Aub, Btn-Aub[R13(me2s)] and Btn-Aub[R15(me2s)] peptides were 90, 87 and 47 RUs, respectively. The protein concentration used for measurement was 40 µm. The binding was performed using BIAcore 3000 in a 20 mm Tris-HCl buffer (ph 8.0) containing 200 mm NaCl, and 0.005% Tween 20. 6

7 Liu et al. Figure S4. Omit difference (Fo-Fc) electron density maps of etud11-aub peptide complex structures. All of the maps were contoured at a cutoff level of 3.5σ. (A) Omit difference map of the Aub[R13(me2s)] complex structure. (B) Omit difference map of the Aub[R15(me2s)] complex structure. (C) Omit map showing the locations of the methyl groups in the Aub[R13(me2s)] complex. The map was generated with the methyl groups omitted during refinement. All maps were generated using the Coot program, and the corresponding refined models were superimposed in a line representation with key residues labeled. A C B 7

8 Figure S5. Representative ITC binding curves of wild-type and N2432A mutant etud11 proteins to Aub peptides: (A) etud11 to R13(me2s); (B) N2432A to R13(me2s); (C) etud11 to R11(me2s); and (D) etud11 to R15(me2s). Top panels, raw titration data in microcalories per second; bottom panels, integrated heat measurements in kilocalories per mole of injected peptide. The horizontal axis indicates the peptide to protein molar ratio. The measurements were carried out at 25 C with 0.3 mm of the etud11 protein in a 20 mm Tris-HCl buffer (ph8.0) containing 150 mm NaCl. Complete fitting parameters are shown in Table S2. 8

9 Table S2: Parameters from ITC data fitting. Bindings to R13(me2s) and R15(me2s) were done in duplicate. The rows highlighted in magenta represent data shown in Fig. S5. Protein Peptide K A (M -1 ) K D ΔH (cal/mol) ΔS (cal/mol/deg) N Chi2/DoF etud11 etud11 N2432A Aub[R13(me2s)] Aub[R13(me2s)] 2.09E4±2.34E3 48±6µM 834.3± ± E4±2.51E3 38±4µM 888.3± ± ± ±0.2mM 1610± ± ±0.2mM 1890± etud11 Aub[R11(me2s)] 1.41E4±1.39E3 71±7µM 521.9± ± etud11 Aub[R15(me2s)] 1.85E5±1.62E4 5.4±0.5µM 768.4± ± E5±1.59E4 5.7±0.5µM 728.9± ±

10 Figure S6. Binding curves of the etud11 protein to Aub[R13(me2s)] and Aub[R15(me2s)] peptides measured by SPR. The immobilize amount of Aub[R13(me2s)] and Aub[R15(me2s)] were 410 and 418 RU, respectively. The curves for decreasing concentrations of protein are shown in the figures from top to bottom. The binding was performed using BIAcore 3000 in a 20 mm Tris-HCl buffer (ph 8.0) containing 200 mm NaCl, and 0.005% Tween 20. The K D values were determined from steady state affinity fitting of the curves using the BIAevaluation software (Biacore). The derived K D values for Aub[R13(me2s)] and Aub[R15(me2s)] binding to etud11 are 36.9 µm and 13.5 µm, respectively. 10