![]() |
![]() |
![]() |
![]() |
![]() |
![]() |
Horizontal Tabs
PDBID |
Structure Details |
Resolution (Å) |
Crystal structure of the N-terminal region of human cohesin subunit STAG1 (Site I, aa 86 – 420) |
2.02 |
|
Crystal structure of the central region of human cohesin subunit STAG1 (Site IV, aa 459 – 915) |
2.31 |
|
Crystal structure of the N-terminal region of human cohesin subunit STAG1 in complex with RAD21 peptide (Site I, aa 86 – 420) |
2.37 |
|
Crystal structure of the central region of human cohesin subunit STAG1 in complex with RAD21 peptide (Site IV, aa 459 – 915) |
3.17 |
Protein Expression and Purification
N-terminal region (Site 1, PDB: 6QB5 and 6RRC)
SGC Construct ID: STAG1A-c087
Protein Region: T86-V420
Vector: pNIC-ZB
Tag: N-terminal 6HIS and ZB tags followed by a TEV protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)):
MHHHHHHSSGVDNKFNKERRRARREIRHLPNLNREQRRAFIRSLRDDPSQSANLLAEAKKLNDAQPKGTENLYFQSMGGTLFEVVKLGKSAMQSVVDDWIESYKQDRDIALLDLINFFIQCSGCRGTVRIEMFRNMQNAEIIRKMTEEFDEDSGDYPLTMPGPQWKKFRSNFCEFIGVLIRQCQYSIIYDEYMMDTVISLLTGLSDSQVRAFRHTSTLAAMKLMTALVNVALNLSIHQDNTQRQYEAERNKMIGKRANERLELLLQKRKELQENQDEIENMMNSIFKGIFVHRYRDAIAEIRAICIEEIGVWMKMYSDAFLNDSYLKYVGWTLHDRQGEVRLKCLKALQSLYTNRELFPKLELFTNRFKDRIVSMTLDKEYDVAVEAIRLVTLILHGSEEALSNEDCENVYHLV
Sequence after tag cleavage:
SMGGTLFEVVKLGKSAMQSVVDDWIESYKQDRDIALLDLINFFIQCSGCRGTVRIEMFRNMQNAEIIRKMTEEFDEDSGDYPLTMPGPQWKKFRSNFCEFIGVLIRQCQYSIIYDEYMMDTVISLLTGLSDSQVRAFRHTSTLAAMKLMTALVNVALNLSIHQDNTQRQYEAERNKMIGKRANERLELLLQKRKELQENQDEIENMMNSIFKGIFVHRYRDAIAEIRAICIEEIGVWMKMYSDAFLNDSYLKYVGWTLHDRQGEVRLKCLKALQSLYTNRELFPKLELFTNRFKDRIVSMTLDKEYDVAVEAIRLVTLILHGSEEALSNEDCENVYHLV
DNA Sequence:
ATGCACCATCATCATCATCATTCTTCTGGTGTGGATAACAAGTTCAACAAGGAGCGTCGAAGAGCTCGCCGTGAAATTCGCCATCTGCCGAACCTGAACCGCGAACAGCGTCGCGCATTTATTCGCAGCCTGCGCGATGATCCGAGCCAGAGCGCGAACCTGCTGGCGGAAGCGAAGAAGCTGAACGATGCGCAGCCGAAGGGTACCGAGAACCTGTACTTCCAATCCATGGGTGGTACATTATTTGAGGTGGTGAAACTGGGGAAAAGTGCAATGCAGTCCGTGGTGGATGACTGGATTGAATCATATAAACAAGACAGGGACATCGCACTTCTGGATTTAATCAACTTTTTTATCCAGTGTTCAGGATGTCGAGGTACTGTGAGAATAGAGATGTTTCGAAATATGCAGAATGCAGAAATCATCAGAAAAATGACTGAAGAATTTGATGAGGACAGTGGTGATTATCCTCTTACCATGCCTGGACCTCAGTGGAAAAAATTTCGTTCAAACTTTTGTGAATTTATTGGAGTCCTGATTCGACAGTGTCAGTATAGCATAATTTATGATGAGTATATGATGGACACAGTAATCTCCCTTTTGACGGGTTTGTCAGACTCCCAGGTCAGAGCTTTTAGGCATACAAGTACCCTGGCTGCCATGAAGCTCATGACTGCTCTGGTGAATGTTGCCTTAAACCTCAGTATTCATCAGGATAATACCCAGAGACAATATGAAGCCGAGAGAAATAAAATGATTGGGAAGAGAGCCAATGAAAGGTTGGAGTTACTACTTCAGAAACGCAAAGAGCTGCAAGAAAATCAGGATGAAATCGAAAATATGATGAACTCTATTTTTAAGGGTATATTTGTTCATAGATACCGTGATGCTATTGCTGAGATTAGAGCCATTTGTATTGAAGAAATTGGAGTATGGATGAAAATGTATAGTGATGCCTTCCTAAATGACAGTTACCTAAAATATGTTGGCTGGACTCTTCATGACAGGCAAGGGGAAGTCAGGCTGAAGTGTTTGAAAGCTCTGCAGAGTCTATATACCAATAGAGAATTATTCCCCAAATTGGAACTATTCACTAACCGATTCAAGGATCGCATTGTATCAATGACACTTGATAAAGAATATGATGTTGCTGTGGAAGCTATTCGATTGGTTACTCTGATACTTCATGGAAGTGAAGAAGCTCTTTCCAATGAAGACTGTGAAAATGTTTACCACTTGGTG
Protein Expression
Medium: Terrific Broth (TB) Merck with 4 ml of glycerol
Antibiotics: Kanamycin
From the glycerol stock, bacteria were inoculated in 15 ml of 1 x TB in a 50 ml tube with Kanamycin 0.05 mg/ml and 0.034 mg/ml of chloramphenicol and grown overnight in a shaker at 37°C, 250rpm. The following day, 4 ml of the overnight culture were inoculated in 1L of TB. The bacteria grew in an incubator at 37°C, shaking 180 rpm. Once the OD reached 2-3, IPTG (300uM) was added to the media and left overnight at 18°C, shaking 180 rpm. The cells were harvested the following day.
Protein Purification
For purification, cell pellets were thawed and resuspended in buffer A (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP)), with the addition of 1x protease inhibitor set VII (Merck, Darmstadt, Germany). Cells were lysed by sonication and cell debris pelleted by centrifugation. Lysates were loaded on to a Ni-sepharose IMAC gravity flow column (GE healthcare), washed with 2 column volumes of wash buffer (buffer A supplemented with 45 mM imidazole), and eluted with 300 mM imidazole in buffer A. The IMAC elution fraction was immediately applied to a 5ml HiTrap SP Sepharose Fast Flow column (GE healthcare), washed with 2 column volumes of elution buffer and eluted with 3 column volumes of 50 mM Hepes pH 7.5, 1 M NaCl, 5 % Glycerol. The purification tag was cleaved with the addition of 1:20 mass ratio of His-tagged TEV protease during overnight dialysis into buffer A. TEV was removed by IMAC column rebinding and final protein purification was performed by size exclusion chromatography using a HiLoad 16/60 Superdex s200 column in buffer A. Protein concentrations were determined by measurement at 280nm (Nanodrop) using the calculated molecular mass and extinction coefficients. Protein masses were checked by LC/ESI-TOF mass spectrometry (Predicted: 39581.2; observed: 39582.3).
Central region (Site IV, PDB:6R7O and 6RRK)
SGC Construct ID: STAG1A-c041
Protein Region: S459-D915
Vector: pNIC28-Bsa4
Tag: N-terminal 6HIS followed by a TEV protease cleavage site
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)):
MHHHHHHSSGVDLGTENLYFQSMSPNGNLIRMLVLFFLESELHEHAAYLVDSLWESSQELLKDWECMTELLLEEPVQGEEAMSDRQESALIELMVCTIRQAAEAHPPVGRGTGKRVLTAKERKTQIDDRNKLTEHFIITLPMLLSKYSADAEKVANLLQIPQYFDLEIYSTGRMEKHLDALLKQIKFVVEKHVESDVLEACSKTYSILCSEEYTIQNRVDIARSQLIDEFVDRFNHSVEDLLQEGEEADDDDIYNVLSTLKRLTSFHNAHDLTKWDLFGNCYRLLKTGIEHGAMPEQIVVQALQCSHYSILWQLVKITDGSPSKEDLLVLRKTVKSFLAVCQQCLSNVNTPVKEQAFMLLCDLLMIFSHQLMTGGREGLQPLVFNPDTGLQSELLSFVMDHVFIDQDEENQSMEGDEEDEANKIEALHKRRNLLAAFSKLIIYDIVDMHAAADIFKHYMKYYNDYGDIIKETLSKTRQID
Sequence after tag cleavage:
SMSPNGNLIRMLVLFFLESELHEHAAYLVDSLWESSQELLKDWECMTELLLEEPVQGEEAMSDRQESALIELMVCTIRQAAEAHPPVGRGTGKRVLTAKERKTQIDDRNKLTEHFIITLPMLLSKYSADAEKVANLLQIPQYFDLEIYSTGRMEKHLDALLKQIKFVVEKHVESDVLEACSKTYSILCSEEYTIQNRVDIARSQLIDEFVDRFNHSVEDLLQEGEEADDDDIYNVLSTLKRLTSFHNAHDLTKWDLFGNCYRLLKTGIEHGAMPEQIVVQALQCSHYSILWQLVKITDGSPSKEDLLVLRKTVKSFLAVCQQCLSNVNTPVKEQAFMLLCDLLMIFSHQLMTGGREGLQPLVFNPDTGLQSELLSFVMDHVFIDQDEENQSMEGDEEDEANKIEALHKRRNLLAAFSKLIIYDIVDMHAAADIFKHYMKYYNDYGDIIKETLSKTRQID
DNA Sequence:
CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGAGCCCGAATGGAAACCTCATTAGGATGCTGGTTCTTTTCTTTCTTGAAAGTGAGTTACATGAACATGCAGCCTACTTGGTGGACAGTTTATGGGAGAGCTCTCAAGAACTGTTGAAAGACTGGGAATGTATGACAGAGTTGCTATTAGAAGAACCTGTTCAAGGAGAGGAAGCAATGTCTGATCGTCAAGAGAGTGCTCTTATAGAGCTAATGGTTTGTACAATTCGTCAAGCTGCTGAGGCACATCCTCCAGTGGGAAGGGGTACCGGCAAGAGAGTGCTAACTGCCAAAGAAAGGAAAACTCAAATTGATGATAGAAACAAATTGACTGAACATTTTATTATTACACTTCCTATGTTACTGTCAAAGTATTCTGCAGATGCAGAGAAGGTAGCAAACTTGCTACAAATCCCACAGTATTTTGATTTAGAAATCTACAGCACAGGTAGAATGGAAAAGCATCTGGATGCTTTATTAAAACAGATTAAGTTTGTTGTGGAGAAACACGTAGAATCAGATGTTCTAGAAGCCTGCAGTAAAACCTATAGTATCTTATGCAGTGAAGAATATACCATCCAGAACAGAGTTGACATAGCTCGAAGCCAGCTGATTGATGAGTTTGTAGATCGATTCAATCATTCTGTGGAAGACCTATTGCAAGAGGGAGAAGAAGCTGATGATGATGACATTTACAATGTTCTTTCTACATTAAAGCGGTTAACTTCTTTTCACAATGCACATGATCTCACAAAATGGGATCTCTTTGGTAATTGCTACAGATTATTGAAGACTGGAATTGAACATGGAGCCATGCCAGAACAGATAGTCGTGCAAGCACTGCAGTGTTCCCATTATTCGATTCTTTGGCAGTTGGTGAAAATTACTGATGGCTCTCCTTCCAAAGAGGATTTGTTGGTATTGAGGAAAACGGTGAAATCCTTTTTGGCTGTTTGCCAGCAGTGCCTGTCTAATGTTAATACTCCAGTGAAAGAACAGGCTTTCATGTTACTCTGTGATCTTCTGATGATTTTCAGCCACCAATTAATGACAGGTGGCAGAGAGGGCCTTCAGCCTTTGGTGTTCAATCCAGATACTGGACTCCAATCTGAACTCCTCAGTTTTGTGATGGATCACGTTTTTATTGACCAAGACGAGGAGAACCAGAGCATGGAGGGTGATGAAGAAGATGAAGCTAATAAAATTGAGGCCTTACATAAAAGAAGGAATCTACTTGCTGCTTTCAGCAAACTTATCATTTATGACATTGTTGACATGCATGCAGCTGCAGACATCTTCAAACACTACATGAAGTATTACAATGACTATGGTGATATTATTAAGGAAACACTGAGTAAAACCAGGCAGATTGATAAAATTCAGTGTGCCAAGACTCTCATTCTCAGTTTGCAACAGTTATTTAATGAACTTGTTCAAGAGCAAGGTTGACAGTAAAGGTGGATACGGATCCGAA
Protein Expression
Medium: Terrific Broth (TB) Merck with 4 ml of glycerol
Antibiotics: Kanamycin
From the glycerol stock, bacteria were inoculated in 15 ml of 1 x TB in a 50 ml tube with Kanamycin 0.05 mg/ml and 0.034 mg/ml of chloramphenicol and grown overnight in a shaker at 37°C, 250rpm. The following day, 4 ml of the overnight culture were inoculated in 1L of TB. The bacteria grew in an incubator at 37°C, shaking 180 rpm. Once the OD reached 2-3, IPTG (300uM) was added to the media and left overnight at 18°C, shaking 180 rpm. The pellet was harvested the following day.
Protein Purification
For purification, cell pellets were thawed and resuspended in buffer A (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 10 mM imidazole, 0.5 mM Tris (2-carboxyethyl) phosphine (TCEP)), with the addition of 1x protease inhibitor set VII (Merck, Darmstadt, Germany). Cells were lysed by sonication and cell debris pelleted by centrifugation. Lysates were loaded on to a Ni-sepharose IMAC gravity flow column (GE healthcare), washed with 2 column volumes of wash buffer (buffer A supplemented with 45 mM imidazole) and eluted with 300 mM imidazole in buffer A. The purification tag was cleaved with the addition of 1:20 mass ratio of His-tagged TEV protease during overnight dialysis into buffer A. TEV was removed by IMAC column rebinding and final protein purification was performed by size exclusion chromatography using a HiLoad 16/60 Superdex s200 column in buffer A. Protein concentrations were determined by measurement at 280nm (Nanodrop) using the calculated molecular mass and extinction coefficients. Protein masses were checked by LC/ESI-TOF mass spectrometry (Predicted: 52692.1; observed: 52693.8).
Structure Determination
6QB5: Crystal structure of the N-terminal region of human cohesin subunit STAG1 (Site I, aa 86 – 420)
Crystallization: For crystallisation the protein was concentrated to 15 mg/ml and crystallised by sitting drop vapor diffusion. Crystals grew in conditions containing 0.1 M Na/K phosphate pH 6.0, 0.2 M NaCl, 34% PEG200. Initial crystals were substantially improved by seeding using a 1000-fold dilution of seed stock. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Data Collection: Data were collected to 2.0 Å resolution at Diamond light source beamline I04-1 and processed using DIALS.
Data Processing: Structures were solved by molecular replacement using the programme PHASER and the structure of the STAG2 SCC1 complex (4PK7) as a search model. The structure was refined using PHENIX REFINE to a final Rfactor = 21.8%, Rfree = 24.5%.
6R7O: Crystal structure of the central region of human cohesin subunit STAG1 (Site IV, aa 459 – 915)
Crystallisation: For crystallisation the protein was concentrated to 5 mg/ml and crystallised by sitting drop vapour diffusion from conditions containing 20 % PEG 3350, 10 % ethylene glycol, 0.2 M sodium malonate, 0.1 M Bis-Tris Propane pH 7.0. Crystals were loop mounted and cryo-cooled by plunging directly into liquid nitrogen.
Data Collection: Data were collected to 2.3 Å resolution at Diamond light source beamline I04-1 and processed using DIALS.
Data Processing: Structures were solved by Molecular replacement using the programme PHASER and the structure of the STAG2 SCC1 complex (4PK7) as a search model. The structure was refined using PHENIX REFINE to a final Rfactor = 22.6 %, Rfree = 26.4 %
6RRC: Crystal structure of the N-terminal region of human cohesin subunit STAG1 in complex with RAD21 peptide (Site I, aa 86 – 420)
Crystallization: For crystallization, a SCC1 peptide corresponding to the sequence KRKLIVDSVKELDSKTIRAQLSDYS was mixed with the protein in a 1:1 ratio and crystallization trials were set up at 12 mg/ml. The peptide bound crystals appeared in conditions containing 2.1 M Ammonium Sulfate, 0.1 M MES pH 6.3. Crystals were transferred to a cryo solution containing well solution supplemented with 25 % Glycerol before being loop mounted and plunged into liquid nitrogen.
Data Collection: Data were collected to 2.4 Å resolution at Diamond light source beamline I04-1, and processed using DIALS.
Data Processing: Structures were solved by Molecular replacement using the programme PHASER and the structure of the STAG2 SCC1 complex (4PK7) as a search model. The structure was refined using PHENIX REFINE to a final Rfactor = 21.4%, Rfree = 24%
6RRK: Crystal structure of the central region of human cohesin subunit STAG1 in complex with RAD21 peptide (Site IV, aa 459 – 915)
Crystallisation: For crystallisation of the Site IV peptide bound crystals, a SCC1 peptide corresponding to the sequence PTKKLMMWKETGGVEKLFSLPAQPLWNNRLLKLFTRCLTP was mixed with the protein in a 1:1 ratio and crystallisation trials were set up at 8.5 mg/ml. Crystals grew from conditions containing 16 % PEG 3350, 0.2 M DL-Malic acid. Crystals were loop mounted and transferred to a cryo solution containing well solution supplemented with 20 % ethylene glycol before being loop mounted and plunged into liquid nitrogen.
Data Collection: Data were collected to 3.2 Å resolution at Diamond light source beamline I24 and processed using DIALS.
Data Processing: Structures were solved by Molecular replacement using the programme PHASER and the structure of the STAG2 SCC1 complex (4PK7) as a search model. The structure was refined using PHENIX REFINE to a final Rfactor = 21.9 %, Rfree = 27.8%
Assays
Alphascreen assay
An alphascreen binding assay was developed using streptavidin alphascreen donor beads and Ni-NTA alphascreen acceptor beads. The assay was performed in 384 well format plates with histidine tagged STAG1A-c041 and a synthetic biotinylated peptide from Rad 21: Biotin-PTKKLMMWKETGGVEKLFSLPAQPLWNNRLLKLFTRCLTPL (residues 356-396). Both protein and peptide were diluted in a buffer containing 25 mM HEPES pH 7.4, 100 mM NaCl, 0.1 % BSA, 0.01 % CHAPS. The assay format was 4µl Buffer + 4µl Protein + 4ul Peptide. Reactions were incubated for 20 minutes and 8 µl of bead mixture was added (1:600 dilution of both beads). The plate was measured after further incubation for 2 hours using a PHERAstar FSX plate reader using excitation and emission wavelengths of 680 and 570 nm.
1. Hill, V. K., Kim, J. S., and Waldman, T. (2016) Cohesin mutations in human cancer. Biochim Biophys WActa 1866, 1-11
2. Benedetti, L., Cereda, M., Monteverde, L., Desai, N., and Ciccarelli, F. D. (2017) Synthetic lethal interaction between the tumour suppressor STAG2 and its paralog STAG1. Oncotarget 8, 37619-37632
3. Mondal, G., Stevers, M., Goode, B., Ashworth, A., and Solomon, D. A. (2019) A requirement for STAG2 in replication fork progression creates a targetable synthetic lethality in cohesin-mutant cancers. Nat Commun 10, 1686
4. van der Lelij, P., Lieb, S., Jude, J., Wutz, G., Santos, C. P., Falkenberg, K., Schlattl, A., Ban, J., Schwentner, R., Hoffmann, T., Kovar, H., Real, F. X., Waldman, T., Pearson, M. A., Kraut, N., Peters, J. M., Zuber, J., and Petronczki, M. (2017) Synthetic lethality between the cohesin subunits STAG1 and STAG2 in diverse cancer contexts. Elife 6
5. Losada, A. (2014) Cohesin in cancer: chromosome segregation and beyond. Nat Rev Cancer 14, 389-393
6. Michaelis, C., Ciosk, R., and Nasmyth, K. (1997) Cohesins: chromosomal proteins that prevent premature separation of sister chromatids. Cell 91, 35-45
7. Canudas, S., and Smith, S. (2009) Differential regulation of telomere and centromere cohesion by the Scc3 homologues SA1 and SA2, respectively, in human cells. J Cell Biol 187, 165-173
8. Hara, K., Zheng, G., Qu, Q., Liu, H., Ouyang, Z., Chen, Z., Tomchick, D. R., and Yu, H. (2014) Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion. Nat Struct Mol Biol 21, 864-870
9. Sakamoto, K. M., Kim, K. B., Kumagai, A., Mercurio, F., Crews, C. M., and Deshaies, R. J. (2001) Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc Natl Acad Sci U S A 98, 8554-8559
10. van der Lelij, P., Newman, J. A., Lieb, S., Jude, J., Katis, V., Hoffmann, T., Hinterndorfer, M., Bader, G., Kraut, N., PEarson, M. A., Peters, J., Zuber, J., Gileadi, O., and Petronczki, M. (2020) STAG1 vulnerabilities for exploiting cohesin synthetic lethality in STAG2-deficient cancers. Life Sci Alliance 28, 3(7)
We respectfully request that this document is cited using the DOI value as given above if the content is used in your work.