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Cloning, expression and purification of human GLP: DNA fragments encoding the methyltransferase domain of human GLP (residues 951-1235), G9a (residues 913-1193), SUV39H2 (residues 112-410) and PRDM2 (residues 2-148) were amplified by PCR and sub-cloned into the pET28a-LIC vector (http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php), downstream of the poly-histidine coding region. The methyltransferase domains of the above proteins were expressed in E.coli BL21 (DE3) codon plus RIL strain (Stratagene) by addition of 1 mM isopropyl-1-thio-D-galactopyranoside and incubated overnight at 15°C. The proteins were purified as following: harvested cells were resuspended in phosphate-buffered-saline (pH 7.4) supplemented with 250 mM NaCl, 2 mM β-mercaptoethanol, 5% glycerol, 0.1% Igepal and 1mM phenylmethyl sulfonyl fluoride. The cells were lysed by passing through a microfluidizer (Microfluidics Corp.). The lysate was loaded onto a 5 ml HiTrap Chelating column (GE Health Care), charged with Ni2+. The column was washed with 10 column volume of 20 mM Tris-HCl (pH 8.0), containing 250 mM NaCl and 50 mM imidazole, 5% glycerol, and the protein was eluted with elution buffer (20 mM Tris-HCl, pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol). The protein was loaded onto a Superdex 200 column (GE Health Care) equilibrated with 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. Thrombin (Sigma) was added to combined fractions containing the target proteins to remove the His6- tag. The protein was further purified to homogeneity by ion-exchange chromatography.
Crystallization: Purified GLP, G9a and SUV39H2 proteins were crystallized in the presence of S-adenosyl-L-homocysteine (Sigma) or S-adenosyl-L-methionine (Sigma) using the hanging drop vapor diffusion method at 20 °C by mixing equal volume of the protein solution with the reservoir solution. The GLP-SAH binary complex (protein/SAH molar ratio of 1:5) was crystallized in 20% PEG 4,000, 10% isopropanol, 0.1 M HEPES (pH 7.5). The G9a-SAH complex (protein/SAH molar ratio of 1:5) was crystallized in 20% PEG 3,350, 0.2 M NaF, 0.1 M Bis-Tris propane (pH 7.5), 5% ethylene glycol. The SUV39H2-SAM complex (protein/SAM molar ratio of 1:10) was crystallized in 20% PEG 10, 000, 0.1 M HEPES (pH 7.5). The GLP-SAH-H3K9me and GLP-SAH-H3K9me2 complexes (protein/SAH/peptide molar ratio of 1:5:10) were crystallized in 16% PEG 4,000, 10% isopropanol, 0.1 M HEPES (pH 7.5). Purified PRDM2 was crystallized using hanging drop vapor diffusion method at 20 °C by mixing 1.5 µl of the protein solution with 1.5 µl of the reservoir solution containing 22% PEG 5,000 MME, 0.2 M ammonium sulfate, 0.1 M MES (pH 7.0). All the crystals were soaked in the corresponding mother liquor supplemented with 20% glycerol as cryoprotectant before freezing in liquid nitrogen, except the G9a-SAH complex crystals, for which paraton-N was used as cryoprotectant.
Data Collection and Structure Determination: X-ray diffraction data were collected at 100K at beamline 17ID of Advanced Photon Source (APS) at Argonne National Laboratory, beamline X25 of the National Synchrotron Light Source, beamline A1 of Cornell High Energy Synchrotron Source (CHESS), Cornell University, and a Rigaku FR-E home source. Data were processed using the HKL-2000 software suite [31]. The structures of methyltransferase domain of GLP, G9a, and SUV39H2 were solved by molecular replacement using the program MOLREP [32]. ARP/wARP [33] was used for automatic model building. Graphics program COOT [34] was used for model building and visualization. PRDM2 structure was solved by single-wavelength anomalous diffraction (SAD) at low resolution, using a seleno-methionine derivative crystal with the program SHELXD [35], and the phasing was performed using SHELXE [36]. The low resolution structure was used as model to solve the native structure at higher resolution. Crystal diffraction data and refinement statistics for the structure are displayed in Supplementary Table 1.
Histone Methyltransferase Assay: The SAHH-coupled assay described by Collazo et al. [37] was optimized and employed to assay the activity of G9a, SETD7 and SETD8. This assay utilizes S-adenosylhomocysteine hydrolase (SAHH) to hydrolyze the methyltransfer product S-adenosylhomocysteine to homocysteine and adenosine in the presence of adenosine deaminase which converts adenosine to inosine. The homocysteine concentration is then determined through conjugation of its free sulfhydryl moiety to a thiol-sensitive fluorophore, ThioGlo (Calbiochem). Assays were performed at room temperature in 25 mM potassium phosphate buffer pH 8, 1 mM EDTA, 2 mM MgCl2 and 0.01% Tween 20. Series of control experiments were conducted to establish the optimum assay condition for each methyltransferase and the optimum conditions were used to determine the kinetic parameters for GLP. Assay cocktails were prepared with 5 µM SAHH to avoid any SAH accumulation while produced from the methyltransferase reaction, 3U/ml of adenosine deaminase from Sigma, 70 µM SAM, and GLP. The peptide concentrations were varied over the range of 2 µM to 4 mM. Assays were initiated by the addition of peptide and immediately after starting the reaction, 2x volume of 20 µM ThioGlo solution was added to each well. The methylation reaction was followed by monitoring the increase in fluorescence using Biotek Synergy2 plate reader with 360/40 nm excitation filter and 528/20 nm emission filter for 20 min in 384 well-plate format. Homocysteine generated in the assay was quantified using standard curves. Activity values were corrected by subtracting background caused by the peptide or the protein. Km and kcat values were calculated using the Michaelis-Menten equation and Sigmaplot 9.0. Standard deviations were calculated from two independent experiments.
Structure superimposition and electrostatic potential coloring: Optimal structure superimpositions were identified with ICM (Molsoft LLC). Briefly, the algorithm uses an iterative procedure to find the best "alignable" main chain core in both structures based on seed alignments of 15 residues as follows: (1) start with the most reliable seed alignment of 15 residues; (2) set all weights to 1; (3) perform weighted superposition and evaluate RMSD; (4) calculate the deviation Di for each backbone atom pair; (5) sort the deviations and find the deviation D50 corresponding to 50-percentile of the deviation array; (6) calculate weights W according to the formula Wi=exp(-D502/Di2); (7) go back to step 3 unless a limit of 10 iterations is reached. The electrostatic potential was calculated with ICM using a boundary element solution of the Poisson equation. Color saturation was set to calculated values of +/- 5 kcal/electron units (+5=blue -5=red) when the electrostatic potential was projected on molecular surfaces.
Note: For further details about these strutcures, please refer to SGC Material and Methods entry for G9a , Suv39H2, GLP, PRDM2.