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Horizontal Tabs
PDB ID |
Structure Details |
Supplier |
2.68 Å structure of FAM83A DUF1669 domain |
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1.58 Å structure of FAM83B DUF1669 domain |
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1.78 Å structure of FAM83B with fragment FM001923a |
Apollo Scientific |
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1.76 Å structure of FAM83B with fragment FM002203a |
Specs |
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1.72 Å structure of FAM83B with fragment FM001730a |
Alfa Aeasar |
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1.80 Å structure of FAM83B with fragment FM001894a |
IBScreen |
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1.84 Å structure of FAM83B with fragment FM000368b |
OxXChem |
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1.81 Å structure of FAM83B with fragment FM002168a |
Key Organics |
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1.74 Å structure of FAM83B with fragment FM001730a |
Alfa Aeasar |
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2.10 Å structure of FAM83B with fragment XS094794b |
IBScreen |
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2.03 Å structure of FAM83B with fragment FM001992a |
IBScreen |
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1.95 Å structure of FAM83B with fragment FM002208b |
Maybridge |
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1.95 Å structure of FAM83B with fragment FF000014a |
Gian Filippo Ruda |
Mass Spectrometry
Protein masses were determined using an Agilent LC/MSD TOF system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% isopropanol in water with 0.1% formic acid. Spectra were analysed using the MassHunter software (Agilent).
Protein Expression and Purification
Human FAM83A DUF1669 domain
Boundaries: residues 122-304
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
FAM83A was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1 litre) in LB medium supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol were incubated at 37°C until OD600 reached 0.8 and then cooled to 18°C and supplemented with 0.4 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) to induce protein expression overnight. Cells were harvested by centrifugation at 5000g, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol) with 0.5 mM TCEP and EDTA-free complete protease inhibitor cocktail (Merck) and flash frozen. On thawing, the resuspended cell pellets were lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 21500 rpm. The His6-tagged protein was immobilised on Ni2+ -Sepharose resin and bound proteins eluted using stepwise gradients of 50-250 mM imidazole in binding buffer. Removal of the His6 tag was performed at 4°C overnight using TEV protease. FAM83A was further purified by size exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer containing 50mM HEPES pH 7.5, 300mM NaCl and 0.5mM TCEP. FAM83A was concentrated using centrifugal ultrafiltration with a 10 kDa molecular weight cut-off point membrane. Purity was confirmed by SDS-PAGE and identify verified by intact mass spectrometry.
Human FAM83B DUF1669 domain
Boundaries: residues 117-294
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
FAM83B was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1 litre) in LB medium supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol were incubated at 37°C until OD600 reached 0.6 and then cooled to 18°C and supplemented with 0.35 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG) to induce protein expression overnight. Cells were harvested by centrifugation at 5000g, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol) with 0.5 mM TCEP and EDTA-free complete protease inhibitor cocktail (Merck) and flash frozen. On thawing, the resuspended cell pellets were lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 21000 rpm. The His6-tagged protein was immobilised on Ni2+ -Sepharose resin and bound proteins eluted using stepwise gradients of 50-250 mM imidazole in binding buffer. Removal of the His6 tag was performed at 4°C overnight using TEV protease. FAM83B was further purified by size exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 0.5 mM TCEP, followed by cation exchange chromatography. FAM83B was loaded onto a HiTrap S 5ml column in a buffer of 50 mM NaCl, 50 mM HEPES pH 7.5 and 0.5 mM TCEP and then eluted using a gradient of 1 M NaCl, 50 mM HEPES pH 7.5 and 0.5 mM TCEP. FAM83B was concentrated using centrifugal ultrafiltration with a 10 kDa molecular weight cut-off point membrane and diluted to a final salt concentration of 220 mM NaCl. Purity was confirmed by SDS-PAGE and identity verified by intact mass spectrometry.
Human FAM83A DUF1669 domain (biotinylated)
Boundaries: residues 122-304
Vector: pNIC-Bio3
Tag and additions: C-terminal biotinylation sequence and TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)-R3-pRARE2-BirA
FAM83A was expressed from the vector pNIC-Bio3 in BL21(DE3)-R3-pRARE2-BirA cells. Cultures (1 litre) in LB medium supplemented with 50 μg/mL kanamycin, 34 μg/mL chloramphenicol and 50 μg/mL streptomycin were incubated at 37°C until OD600 reached 0.6 and then cooled to 18°C and supplemented with 0.4 mM IPTG and 100 µM biotin solution to induce biotinylated protein expression overnight. In the morning cultures were supplemented with a further 100 µM biotin solution and incubated for an hour before harvesting. Cells were harvested by centrifugation at 5000g, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol, 0.5mM TCEP) with EDTA-free complete protease inhibitor cocktail (Merck) and flash frozen. On thawing, the resuspended cell pellets were lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 21500 rpm. The His6-tagged protein was immobilised on Ni2+ -Sepharose resin and bound proteins eluted using stepwise gradients of 50-250 mM imidazole in binding buffer. Removal of the His6 tag was performed at 4°C overnight using TEV protease. FAM83A was further purified by size exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 0.5 mM TCEP. FAM83A was concentrated using centrifugal ultrafiltration with a 3 kDa molecular weight cut-off point membrane. Purity was confirmed by SDS-PAGE and identify and biotinylation verified by intact mass spectrometry.
Human FAM83B DUF1669 domain (biotinylated)
Boundaries: residues 117-294
Vector: pNIC-Bio3
Tag and additions: C-terminal biotinylation sequence and TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)-R3-pRARE2-BirA
FAM83B was expressed from the vector pNIC-Bio3 in BL21(DE3)-R3-pRARE2-BirA cells. Cultures (1 litre) in LB medium supplemented with 50 μg/mL kanamycin, 34 μg/mL chloramphenicol and 50 μg/mL streptomycin were incubated at 37°C until OD600 reached 0.6 and then cooled to 18°C and supplemented with 0.4 mM IPTG and 100 µM biotin solution to induce biotinylated protein expression overnight. In the morning cultures were supplemented with a further 100 µM biotin solution and incubated for an hour before harvesting. Cells were harvested by centrifugation at 5000g, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol, 0.5 mM TCEP) with EDTA-free complete protease inhibitor cocktail (Merck) and flash frozen. On thawing, the resuspended cell pellets were lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 21500 rpm. The His6-tagged protein was immobilised on Ni2+ -Sepharose resin and bound proteins eluted using stepwise gradients of 50-250 mM imidazole in binding buffer. Removal of the His6 tag was performed at 4°C overnight using TEV protease. FAM83B was further purified by size exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 0.5 mM TCEP. FAM83B was concentrated using centrifugal ultrafiltration with a 3 kDa molecular weight cut-off point membrane. Purity was confirmed by SDS-PAGE and identify and biotinylation verified by intact mass spectrometry.
Human CSNK1D
Boundaries: residues 1-294
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
CSNK1D was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1 litre) in LB medium supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol were incubated at 37°C until OD600 reached 0.6 and then cooled to 18°C and supplemented with 0.4 mM IPTG to induce protein expression overnight. Cells were harvested by centrifugation at 5000g, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol, 0.5 mM TCEP) with EDTA-free complete protease inhibitor cocktail (Merck) and flash frozen. On thawing, the resuspended cell pellets were lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 21000 rpm. The His6-tagged protein was immobilised on Ni2+ -Sepharose resin and bound proteins eluted using stepwise gradients of 50-250 mM imidazole in binding buffer. Removal of the His6 tag was performed at 4°C overnight using TEV protease. CSNK1D was further purified by size exclusion chromatography using a Superdex 200 16/60 column (GE Healthcare) equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl and 0.5 mM TCEP before concentration using centrifugal ultrafiltration with a 10 kDa molecular weight cut-off point membrane. Purity was confirmed by SDS-PAGE and identify verified by intact mass spectrometry.
Human CSNK1E
Boundaries: residues 1-294
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
CSNK1E was expressed in E. coli strain BL21(DE3) R3-pRARE2 using the pNIC28-Bsa4 vector, which encodes for a N-terminal hexahistidine (6XHis) tag and TEV cleavage site. Cultures were grown at 37°C in LB medium supplemented with 50 μg/mL kanamycin and 34 μg/mL chloramphenicol to an OD of 0.6, before expression at 18°C overnight by induction with 0.4 mM isopropyl 1-thio-β-D-galactopyranoside. Cells were harvested by centrifugation at 5000 g and pellets resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole) supplemented with Calbiochem protease inhibitor set III. Cells were lysed by sonication before clarification of the lysate by centrifugation in a JA 25.50 rotor at 36,000 g. His-tagged proteins were immobilized on Ni-sepharose and bound proteins were eluted using step gradients of imidazole (50-250 mM). CSNK1E protein was cleaved with TEV protease overnight at 4°C and purified further by size exclusion chromatography using an S75 HiLoad 16/60 Superdex column equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Protein was concentrated by centrifugal ultrafiltration using a 3 kDa molecular weight cut-off concentrator. Protein concentration was determined by measuring absorbance at 280 nm. Protein purity of >95% was confirmed by SDS-PAGE and construct identity and tag cleavage were verified by mass spectrometry.
Structure Determination
FAM83A (PDB: 4URJ, 2.68 Å)
FAM83A DUF1669 domain (a.a 122-304) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl and concentrated to 12 mg/mL. Crystals were grown at 20°C in 150 nL sitting drops at a 1:2 protein: reservoir solution comprising 0.8 M sodium phosphate monobasic, 0.8 M potassium phosphate dibasic and 0.1 M HEPES pH 7.5. Before mounting, crystals were cryoprotected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I03. Diffraction data were processed using XDS (16) and molecular replacement was performed using PHENIX.MR_ROSETTA (17). Model building and refinement were done using COOT (18) and PHENIX (19).
FAM83B (PDB: 5LZK, 1.58 Å)
FAM83B DUF1669 domain (a.a 117-294) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl and concentrated to 12 mg/ml. Crystals were grown at 4°C in 150 nL sitting drops at a 1:1 protein: reservoir solution comprising 30.45% PEG3350, 0.2 M sodium iodide, 0.1 M bis-tris-propane pH 9.1 and 10% ethylene glycol. Before mounting, crystals were cryoprotected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04. Diffraction data were processed using XDS (16) and molecular replacement was performed using PHASER (20) using 4URJ as the starting model. Model building and refinement were done using COOT (18) and PHENIX (19).
FAM83B Fragment screening (PDBs: 5HQI, 5HQJ, 5HQK, 5HQL, 5HQM, 5HQN, 5HQO, 5HQP, 5HQQ, 5HQR)
P21 crystal form: FAM83B DUF1669 domain (a.a 117-294) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl and concentrated to 13.1 mg/mL. Crystals were grown at 4°C in 300 nL sitting drops at a 1:2 protein: reservoir solution comprising 29% PEG3350, 0.1 M bis-tris-propane pH 8, 0.2 M NaI and 10% ethylene glycol. Fragments belonging to the DSPL1 library (500 mM in DMSO) were soaked at 20% fragment to drop volume for approximately 1 hour prior to addition of 20% ethylene glycol. Crystals were fished and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04-1. Data was processed using either Xia2 (21) or autoPROC (22). Data was phased using DIMPLE (CCP4 suite (23)) and bound fragments identified using PanDDA (13) in the context of the XChemExplorer software suite. Bound fragments were refined using REFMAC (14).
P4122 crystal form: FAM83B DUF1669 domain (a.a 117-294) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl and concentrated to 12 mg/mL. Crystals were grown at 20°C in 600 nL sitting drops at a 2:3 protein: reservoir solution comprising 0.15 M NaCl and from 28.7-31.6% tacsimate. Fragments belonging to the DSPL2 and OxXchem libraries (500 mM in DMSO) were soaked at 20% fragment to drop volume for approximately 1 hour prior to addition of 20% ethylene glycol. Crystals were fished and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04-1. Data was processed using either Xia2 (21) or autoPROC (22). Data was phased using DIMPLE (CCP4 suite (23)) and bound fragments identified using PanDDA (13) in the context of the XChemExplorer software suite. Bound fragments were refined using REFMAC (14).
FAM83B follow up fragment screening and structure determination (PDB: 5HQS, 1.95 Å)
FAM83B DUF1669 domain (a.a 117-294) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl and concentrated to 12 mg/mL. Crystals were grown at 20°C in 600 nL sitting drops at a 2:3 protein: reservoir solution comprising 0.15 M NaCl and from 28.7-31.6% tacsimate. Fragment follow-up compounds (200 mM in DMSO) were combined with mother liquor and ethylene glycol in a ratio of 1:7:2 and soaked for approximately 1 hour. Diffraction data were collected at Diamond Light Source beamline I04-1. Data was processed using either Xia2 (21) or autoPROC (22). Data was phased using DIMPLE (CCP4 suite (23)) and bound fragments identified using PanDDA (13) in the context of the XChemExplorer software suite. Bound fragments were refined using REFMAC (14).
Assays
Evaluation of fragment binding to FAM83B by SPR
SPR experiments were performed in 25 mM HEPES pH 7.5, 150 mM NaCl, 0.05% Tween-20, 2% DMSO. Biotinylated FAM83B and FAM83A were immobilized to approximately 6000 RU on streptavidin SPR chips (GE Healthcare) using a Biacore S200 surface plasmon resonance analyser. Fragments with a maximum concentration of 1 mM diluted in running buffer were injected for 15 seconds at 30 µL/min and the responses were recorded. The responses at 10 seconds after injection were analysed. The effects of any potential mismatch between the DMSO in the running buffer and that used for fragment dilution was corrected for.
Evaluation of casein kinase binding to FAM83B by Biolayer Interferometry (BLI)
BLI experiments used a buffer comprising 25 mM HEPES pH 7.5, 100 mM NaCl, 0.05% Tween-20, 0.1% BSA. Biotinylated FAM83B was immobilized onto streptavidin Octet sensors until saturation was observed. The sensors were then washed and dipped into wells of casein kinases at various concentrations up to 25 µM and the BLI signal recorded using an Octet RED384 system.
In vitro pull down assay for FAM83A binding to CSNK1E.
For in vitro pull down assay, all proteins and Ni-sepharose were equilibrated in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole) prior to use. 300 μg 6xHis-FAM83A (a.a. 122-304) was immobilised onto 200 μl Ni-sepharose and washed before addition of 100 μg CSNK1E. The Ni-sepharose was then washed with binding buffer and the flow through collected. Two 1 mL wash steps were performed using binding buffer before bound proteins were eluted with 1 mL binding buffer supplemented with 250 mM imidazole. Fractions were run on a SDS-PAGE gel alongside the original protein solutions for molecular weight reference.
Other cellular and in vivo assays
Other cellular and in vivo assays are published (11,12).
- Bartel, C. A., Parameswaran, N., Cipriano, R., and Jackson, M. W. (2016) FAM83 proteins: Fostering new interactions to drive oncogenic signaling and therapeutic resistance. Oncotarget 7, 52597-52612
- Cipriano, R., Miskimen, K. L., Bryson, B. L., Foy, C. R., Bartel, C. A., and Jackson, M. W. (2014) Conserved oncogenic behavior of the FAM83 family regulates MAPK signaling in human cancer. Mol Cancer Res 12, 1156-1165
- Snijders, A. M., Lee, S. Y., Hang, B., Hao, W., Bissell, M. J., and Mao, J. H. (2017) FAM83 family oncogenes are broadly involved in human cancers: an integrative multi-omics approach. Mol Oncol 11, 167-179
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- Cipriano, R., Graham, J., Miskimen, K. L., Bryson, B. L., Bruntz, R. C., Scott, S. A., Brown, H. A., Stark, G. R., and Jackson, M. W. (2012) FAM83B mediates EGFR- and RAS-driven oncogenic transformation. J Clin Invest 122, 3197-3210
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