Expanded View Figures

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1 EMO reports rystal structure of Mis18 Yippee-like domain Lakxmi Subramanian et al Expanded View Figures Figure EV1. Structural characterization of the N-terminal Yippee-like globular domain of spmis18. Stereo image of electron density map (2F o F c, contoured at 1r) corresponding to b-sheet I. Structural superposition of spmis onto its closest structural homologs, ereblon TD (PD: 4TZ), RIG-I (PD: 2QFD), Mss4(PDD: 1FWQ), and Msr (PD: 3E0O). Structural superpositions were carried out using PDefold web server ( Structural superposition of spmis with its structural homologs bound to their substrates. For clarity, structural homologs of spmis are not shown. D Surface representation of spmis where the cradle-shaped pocket and amino acid residues mutated in the complementation assay (Fig 1E) are highlighted. EV1 EMO reports ª 2016 The uthors

2 Lakxmi Subramanian et al rystal structure of Mis18 Yippee-like domain EMO reports K110 K110 Q30 Q30 V32 V32 F29 F29 Q112 Q112 S35 Y114 S35 Y114 P20 P20 Q19 Q19 N118 N118 ereblon TD (PD:4TZ) RIG-I (PD:2QFD) Mss4 (PD:1FWQ) Msr (PD:3E0O) D Y74 Y90 S107 T Thalidomide - ereblon RN - RIG-I Peptide - Msr Figure EV1. ª 2016 The uthors EMO reports EV2

3 EMO reports rystal structure of Mis18 Yippee-like domain Lakxmi Subramanian et al Figure EV2. Evaluation of the dimerization ability of Yippee-like domains of Mis18 proteins. SE-MLS profiles of His-spMis (left panel) and His-spMis I31 (right panel). Refractive index (RI, left y-axis) and molar mass (MW, right y-axis) profiles show that His-spMis (predicted MW of a monomer: 16.2 ) is a dimer (measured MW: 33.3 ) and His-spMis I31 (predicted MW of monomer: 16.2 ) is a monomer (measured MW: 15.6 ). Native PGE analysis of dimer interface II mutants. Interface mutants I31, V22E, Y114E, and Y114 migrated faster than wt spmis , confirming the effect these mutations have on the oligomeric structure of spmis SE-MLS profiles of His-hsMis18a (top panel), His-hsMis18b (middle panel), and His-hsMis18a His-hsMis18b (bottom panel). While HishsMis18a (predicted MW of a monomer: 14.7 ) formed a homodimer (measured MW: 31.3 ), His-hsMis18b (predicted MW of a monomer: 13.6 ) remained as a monomer (measured MW: 15.0 ). His-hsMis18a His-hsMis18b (predicted MW of a monomer: 28.3 ) formed a heterodimer (measured MW: 27.5 ). D SDS PGE analysis of Ni-NT pull-down experiment, where His-hsMis18a and GST-hsMis18b were co-expressed in E. coli as wt or dimer-disrupting mutant proteins. While the GST pull-down assay shown in Fig 2E confirmed the inability of GST-hsMis18b V77E/Y172D to interact with His-hsMis18a V82E/ Y176D, the corresponding Ni-NT pull-down shown here confirms the abundant expression of His-hsMis18a V82E/Y176D in the input. We note that His-hsMis18a V82E/Y176D migrates as a doublet. Western blot analysis (data not shown) using anti-his antibody confirmed the presence of His-tag on both bands suggesting the unstable nature of His-hsMis18a V82E/Y176D mutant (possibly due to its inability to homo- or heterodimerize). EV3 EMO reports ª 2016 The uthors

4 Lakxmi Subramanian et al rystal structure of Mis18 Yippee-like domain EMO reports I31 I31 V22E Y114E Y114 hsmis18α hsmis18β D His-hsMis18α His-hsMis18αα V82E/Y176D GST-hsMis18β GST-hsMis18β V77E/Y172D His-Pulldown hsmis18α hsmis18β GST-hsMis18β His-hsMis18α Figure EV2. ª 2016 The uthors EMO reports EV4

5 EMO reports rystal structure of Mis18 Yippee-like domain Lakxmi Subramanian et al Figure EV3. haracterization of the overall oligomeric state of spmis18 fl. SE profile and respective SDS PGE analysis of fractions (bottom panel) for His-GFP-spMis18 fl. Superose 610/300 column was used for His-GFP-spMis18 fl. Multiple sequence alignment of spmis18 with its orthologs highlights the presence of a low-complexity region (Lys/rg-rich region) at the extreme -terminus unique to spmis18., D SE profiles (top panels) and respective SDS PGE analyses of their fractions (bottom panels) for His-spMis18D and His-GFP-spMis18 -term-a, respectively. Superdex 200 increase 10/300 column was used for His-spMis18D and His-GFP-spMis18 -term-a. EV5 EMO reports ª 2016 The uthors

6 Lakxmi Subramanian et al rystal structure of Mis18 Yippee-like domain EMO reports His-GFP-spMis18 fl ** * * ** * * ** ** Low omplexity Region D His-GFP-spMis18 -term-α His-spMis Figure EV3. ª 2016 The uthors EMO reports EV6

7 EMO reports rystal structure of Mis18 Yippee-like domain Lakxmi Subramanian et al GFP np1 DPI Merge spmis18 fl -GFP spmis18 fl I31-GFP spmis18 fl Y114-GFP wt thiamine - thiamine spmis18 spmis18 fl I31 spmis18 fl Y114 % ppt cc2 DN ENP- np1 hip Induced Repressed 10 0 no tag spmis18 fl spmis18 fl I31 spmis18 fl Y114 Figure EV4. Ectopically expressed spmis18 fl proteins localize to the nucleus and do not alter cell growth. Ectopically expressed spmis18 fl -GFP localizes throughout the nucleus in a MeDiY dimerization-independent manner. Immunofluorescence of wild-type S. pombe cells ectopically expressing GFP-tagged spmis18 fl (wt or mutants), stained with antibodies to GFP (green) and ENP- np1 (red), and DPI (blue). Ectopic expression of dimer II interface mutants does not affect growth of wild-type S. pombe cells. Fivefold serial dilutions of cells expressing the indicated spmis18 fl constructs integrated at the leu1 locus in the genome, spotted on complete PMG phloxine media supplemented with (repressed) or without (expressed) thiamine, and incubated at the indicated temperatures; dead cells stain dark pink. Mutations that disrupt MeDiY dimerization cause no significant change in ENP- np1 association with centromeres in wild-type cells. qhip analyses of ENP- np1 association with centromere 2 (cc2) in the indicated strains when grown in complete PMG media supplemented with (repressed) or without (expressed) thiamine. Error bars represent standard deviation between at least three biological replicates. EV7 EMO reports ª 2016 The uthors