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Supporting Information Supporting Information Liban et al. 10.1073/pnas.1619170114 SI Materials and Methods X-Ray Structure Determination. Data were collected from single Protein Expression and Purification. Human DP1 (residues 199 to crystals under cryogenic conditions (100 K) at the Advanced Light 350) and E2F4 (residues 91 to 198) or E2F5 (residues 124 to 232) Source (beamline 8.3.1) and Advanced Photon Source (APS) proteins were coexpressed from pET-derived vectors containing (beamline 23IDB). For E2F4–DP1, a molecular replacement so- N-terminal GST and N-terminal 6×His tags, respectively. Escherichia lution was found with Phaser (37) using PDB ID code 2AZE as a coli BL21(DE3) cells cotransformed with both vectors were grown search model. We could not find a molecular replacement solu- – – to an OD600 of 0.6 to 0.8 and induced with 0.5 mM isopropyl β-D- tion for p107C E2F5 DP1 native diffraction data, perhaps be- 1-thiogalactopyranoside (IPTG), with protein expression taking cause of the different angles between the coiled-coil and marked- place overnight at 18 °C. Cells were pelleted at 3,470 × g for box domains (Fig. 2B). Phases were alternatively calculated from 10 min and resuspended in a lysis buffer containing 25 mM Hepes, a single anomalous diffraction (SAD) experiment using a sele- 100 mM NaCl, 5 mM DTT, 5 mM EDTA, 10% glycerol, and nomethionine derivative. SAD data were collected at the sele- 1 mM PMSF (pH 8). A cell homogenizer was used to lyse the cells nium peak energy. Six selenium sites and an initial structural and, following centrifugation at 25,200 × g for 30 min, the model were generated by using the program AutoSol (38) from resulting soluble fraction was purified using glutathione Sepharose within the Phenix (39) interface. Although we expected in- affinity chromatography followed immediately by Ni Sepharose corporation of 12 selenium atoms (6 per molecule in the asym- affinity chromatography. Following the affinity chromatography metric unit), only 8 methionine residues were visible in the columns, the eluate was further purified by anion-exchange chro- structure. High-resolution native data and the initial model from matography and cleaved with GST–TEV (Tobacco Etch Virus) pro- AutoSol were then used for further model building and re- tease overnight at 4°C. To remove cleaved GST and His tags, proteins finement by using Coot (40) and Phenix, respectively. Coordi- CM were again passed over glutathione and nickel Sepharose resin, and nates and structure factors for the E2F4–DP1 and p107C– CM finally the heterodimer was subjected to size-exclusion chromatog- E2F5–DP1 structures were deposited in the Protein Data raphy to achieve the final pure sample. All p107C and RbC con- Bank under ID codes 5TUU and 5TUV, respectively. structs were expressed and purified similar to as described (19). Isothermal Titration Calorimetry. Preceding ITC experiments, Protein Phosphorylation. Phosphorylation of p107C peptides was protein samples were dialyzed overnight at 4 °C in 20 mM Tris, achieved by adding 10% by mass Cdk2–Cyclin A to the p107C 100 mM NaCl, and 1 mM beta-mercaptoethanol. Binding experi- substrate in 25 mM Tris, 150 mM NaCl, 5 mM DTT, 10 mM ments involving p107C were conducted at pH 8.0, and experiments MgCl2, and 1 mM ATP. The kinase was purified away from involving RbC were performed at pH 7.0. Typically, 0.5 to 1 mM phosphorylated p107C using size-exclusion chromatography. peptide was injected into 20 to 40 μMDP1–E2FCM using a Incorporation of phosphate was determined quantitatively using MicroCal VP-ITC. Origin software (www.originlab.com) was used electrospray mass spectrometry. Samples were desalted and di- to calculate binding constants by fitting the data to a one-site rectly injected into the spectrometer. binding model. The error associated with the reported binding constants is the SD calculated from two to four independent Protein Crystallization. Before crystallization trials, the E2F4– binding experiments. DP1CM heterodimer was purified over an SD75 Superdex column in 20 mM Tris (pH 8.0), 100 mM NaCl, and 4 mM Tris(2- Homolog Detection and Phylogenetic Analyses. We used the HMMER carboxyethyl)phosphine (TCEP). Crystal trays were set at 13 mg/mL, 3 (41) package and profile-hidden Markov models (profile-HMMs) using a 1:1 protein-to-buffer ratio at 18 °C. Diffraction-quality built previously for eukaryotic-wide phylogenetic analyses (35) to crystals appeared within 3 to 4 d in 100 mM Mes, 100 mM NaCl, retrieve homologs (E-value threshold of 1E-10) of the E2F–DP and 16% PEG 6000 (pH 6.3). Crystals were harvested, incubated and pocket protein family from 52 genomes with particular in the above condition plus 25% ethylene glycol as a cryoprotec- focus on the Metazoa. Some detected homologs were discarded tant, and flash-frozen in liquid nitrogen before data collection. because they were incomplete or too divergent to be included To crystallize the trimeric protein complex, p107C was mixed in a in our phylogenies. All reliable homologs were aligned using 2:1 molar ratio with purified DP1–E2F5. The complex was isolated MAFFT-L-INS-i (-maxiterate 1000) (42). Resulting alignments using an SD75 Superdex column equilibrated in 20 mM Hepes were masked using probabilistic alignment masking with ZORRO (pH 7.0), 100 mM NaCl, and 4 mM TCEP. Crystal trays were set (43). ProtTest 3 (44) was used to determine the empirical amino at 15 mg/mL and incubated at 18 °C. Diffracting crystals were acid evolutionary model that best fit each of our protein datasets grown in 100 mM Hepes (pH 7.0), 7% PEG 5000, 5% 1-propanol, using several criteria: Akaike information criterion, corrected and 2% 2-propanol. Crystals were flash-frozen in liquid nitrogen Akaike information criterion, Bayesian information criterion, and in a buffer containing the crystallization condition supplemented decision theory (E2F–DP: JTT+I+G+F; Rb: LG+I+G+F). Last, with 25% glycerol. Both native and selenomethionine-containing for each dataset (Datasets S1 and S2) and its best-fitting model, crystals were grown using this process. All crystals in this study we ran different phylogenetic programs that use maximum-likelihood were grown using the sitting-drop vapor-diffusion method. methods with different algorithmic approximations (RAxML To incorporate selenomethionine into E2F5–DP1CM protein and PhyML) to reconstruct the phylogenetic relationships be- complexes, cotransformed BL21 cells were grown in M9 minimal tween proteins. For RAxML (45) analyses, the best-likelihood media to an OD600 of 0.6. The methionine pathway was inhibited tree was obtained from five independent maximum-likelihood through addition of lysine, phenylalanine, and threonine at runs started from randomized parsimony trees using the empirical 100 mg/L and isoleucine and valine at 50 mg/L, and supplemented evolutionary model provided by ProtTest. We assessed branch with 100 mg of selenomethionine per L. Twenty minutes after support via rapid bootstrapping (RBS) with 100 pseudoreplicates. addition of the amino acids, cells were induced with 0.5 mM PhyML 3.0 (46) phylogenetic trees were obtained from five in- IPTG. E2F5–DP1CM containing selenomethionine was otherwise dependent randomized starting neighbor-joining trees using the expressed and purified similar to unlabeled protein. best topology from both NNI (nearest neighbor interchanges) Liban et al. www.pnas.org/cgi/content/short/1619170114 1of11 and SPR (subtree pruning and regrafting) moves. Nonpara- independent PhyML 3.0 runs. The Rb phylogeny (Fig. S6) and metric Shimodaira–Hasegawa–like approximate-likelihood E2F phylogeny (Fig. S7) were then used to classify orthologs and ratio tests (SH-aLRTs) and parametric à la Bayes aLRTs paralogs to create Fig. S5. Taxa silhouettes in Fig. 5 were obtained (aBayes) were calculated to determine branch support from two from www.phylopic.org. p107 CTD Construct 994- 994- 1000- 1000- --- MW 1038 1031 1038 1031 (kDa) U BUBUBUBUB 116/97 66 45 31 GST-p107 21.5 14.4 DP1 E2F4 Fig. S1. Coprecipitation assay identifying a minimal p107 CTD fragment that binds E2F4–DP1CM. One hundred micrograms of the indicated GST– p107 fragment and 100 μg of purified E2F4–DP1CM were incubated on ice for 1 h in a reaction volume of 200 μL containing 100 mM NaCl, 25 mM Tris, and 5 mM DTT (pH 8.0). Proteins were affinity-precipitated with glutathione Sepharose resin, washed, and eluted in the binding buffer plus 10 mM glutathione. For each binding reaction, the eluate containing bound proteins (B) was loaded onto an SDS polyacrylamide gel along with a sample of the unbound (U) reaction. Liban et al. www.pnas.org/cgi/content/short/1619170114 2of11 AB E2F1 E2F1 E2F4 E2F5 DP1 DP1 CD E2F4 E2F5 E2F5 E2F5 DP1 DP1 E E2F DP1 90° β5 β5 Fig. S2. Conservation of the E2F–DP CM structure. Comparison of CM β-sandwich domains reveals similar structures. (A) Alignment of E2F4–DP1CM and E2F1– DP1CM structures. The root-mean-square deviation (rmsd) of the Cα position is 0.77 Å. (B) Alignment of E2F5–DP1CM and E2F1–DP1CM structures. Rmsd is 1.24 Å. (C) Alignment of E2F5–DP1CM and E2F4–DP1CM structures. Rmsd is 1.35 Å. (D) Alignment of two different E2F5–DP1CM heterodimers in the asymmetric unit of E2F5–DP1–p107C crystals. Rmsd is 1.14 Å. (E) E2F4–DP1CM is shown with residues that are conserved among E2F paralogs in yellow. For sequence alignment, see Fig. 1B. Most of the 20 residues that are identical among family members are critical for the structural core of the domain. A group of highly conserved residues forms the last E2F strand (β5) and the preceding loop. Their side chains form a surface on the edge of the sandwich opposite the edge that binds pocket proteins (Fig.
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