Human tumor protein p73

Target ID:

TP73A

Aliases:

P73TP73

Deposition Date:

Wednesday 30th June 2010

Families:

Miscellaneous

Related Diseases:

CancerSignallingNeurobiology

Structure type:

apo

Download Coordinates:

2XWC

Authors:

P. Canning, Y. Zhang, M. Vollmar, T. Krojer, E. Ugochukwu, J.R.C. Muniz, F. Von Delft, J. Weigelt, C.H. Arrowsmith, A.M. Edwards, C. Bountra, A. Bullock

Site:

Oxford

ICM Datapack:

ICM Datapack

2XWC

tabs

Structure Details

p73 belongs with p53 and p63 to the p53 family of transcription factors. The different family members exhibit high sequence identity among their transactivation (TA), DNA-binding (DBD), and tetramerization (ISO) domains and can interact to regulate the same target promoter (Danilova et al., 2008), (Murray-Zmijewski et al., 2006). Two different promoters in the p73 gene give rise to DeltaN or TA isoforms either lacking or containing an N-terminal transactivation domain, respectively. In addition, alternative splicing produces different C-termini, resulting in 29 different splice forms. Extended C-terminal isoforms of both p63 and p73 contain a SAM domain that is absent in p53. Depending on their domain composition, the different isoforms can act either as transcriptional activators or repressors. Isoforms harbouring the transactivation domain mostly suppress cell growth, while those lacking the domain promote it (Murray-Zmijewski et al., 2006).

Knockout mice for all p73 isoforms displayed hippocampal dysgenesis revealing unique functions for p73 in brain development. The mice showed a defective fluid system in the respiratory airways and brain exemplified by the development of hydropcephalus. Moreover, the mice developed chronic infections and inflammation, as well as abnormalities in their pheromone sensory pathways resulting in abnormal reproductive and social behaviour (Yang et al., 2000) (Yang and McKeon, 2000). The explicit functions for the isoforms lacking the N-terminus were revealed by the specific phenotypes of these knockout mice. Knockout mice for DeltaNp73 displayed signs of neurodegeneration with atrophic choroid plexuses and reduced neurons in other brain areas (Wilhelm et al.). In contrast, knockout mice for TAp73 isoforms showed genomic instability, were infertile and produced oocytes exhibiting spindle abnormalities as well as having a high incidence of spontaneous tumors (Tomasini et al., 2009).

p73 is differentially expressed in a number of tumours. Depending on the cellular context and isoform expressed, p73 can act either as a tumor suppressor or have oncogenic potential (Rosenbluth and Pietenpol, 2008) (Zawacka-Pankau et al.). The p73 G4C14-to-A4T14 polymorphism has been suggested to be associated with risk for gastric cancer, squamous cell carcinoma of head and neck and/or outcome of preoperative radiotherapy in rectal cancer patients (De Feo et al., 2009) (Li et al., 2009) (Loof et al., 2009). Additionally, GWAS implicated p73 in chronic rhinosinusitis (Tournas et al.).

Here we present the structure of the p73 DNA binding domain refined at 1.80 Å resolution.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:40125802

SGC Construct ID: TP73A-c009

GenBank GI number: gi|4885645

Vector: pNIC-CTHF. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
AGGGGACGCAGCGAAACCGGGGCCCGCGCC
AGGCCAGCCGGGACGGACGCCGATGCCCGG
GGCTGCGACGGCTGCAGAGCGAGCTGCCCT
CGGAGGCCGGCGTGGGGAAGATGGCCCAGT
CCACCGCCACCTCCCCTGATGGGGGCACCA
CGTTTGAGCACCTCTGGAGCTCTCTGGAAC
CAGACAGCACCTACTTCGACCTTCCCCAGT
CAAGCCGGGGGAATAATGAGGTGGTGGGCG
GAACGGATTCCAGCATGGACGTCTTCCACC
TGGAGGGCATGACTACATCTGTCATGGCCC
AGTTCAATCTGCTGAGCAGCACCATGGACC
AGATGAGCAGCCGCGCGGCCTCGGCCAGCC
CCTACACCCCAGAGCACGCCGCCAGCGTGC
CCACCCACTCGCCCTACGCACAACCCAGCT
CCACCTTCGACACCATGTCGCCGGCGCCTG
TCATCCCCTCCAACACCGACTACCCCGGAC
CCCACCACTTTGAGGTCACTTTCCAGCAGT
CCAGCACGGCCAAGTCAGCCACCTGGACGT
ACTCCCCGCTCTTGAAGAAACTCTACTGCC
AGATCGCCAAGACATGCCCCATCCAGATCA
AGGTGTCCACCCCGCCACCCCCAGGCACTG
CCATCCGGGCCATGCCTGTTTACAAGAAAG
CGGAGCACGTGACCGACGTCGTGAAACGCT
GCCCCAACCACGAGCTCGGGAGGGACTTCA
ACGAAGGACAGTCTGCTCCAGCCAGCCACC
TCATCCGCGTGGAAGGCAATAATCTCTCGC
AGTATGTGGATGACCCTGTCACCGGCAGGC
AGAGCGTCGTGGTGCCCTATGAGCCACCAC
AGGTGGGGACGGAATTCACCACCATCCTGT
ACAACTTCATGTGTAACAGCAGCTGTGTAG
GGGGCATGAACCGGCGGCCCATCCTCATCA
TCATCACCCTGGAGATGCGGGATGGGCAGG
TGCTGGGCCGCCGGTCCTTTGAGGGCCGCA
TCTGCGCCTGTCCTGGCCGCGACCGAAAAG
CTGATGAGGACCACTACCGGGAGCAGCAGG
CCCTGAACGAGAGCTCCGCCAAGAACGGGG
CCGCCAGCAAGCGTGCCTTCAAGCAGAGCC
CCCCTGCCGTCCCCGCCCTTGGTGCCGGTG
TGAAGAAGCGGCGGCATGGAGACGAGGACA
CGTACTACCTTCAGGTGCGAGGCCGGGAGA
ACTTTGAGATCCTGATGAAGCTGAAAGAGA
GCCTGGAGCTGATGGAGTTGGTGCCGCAGC
CACTGGTGGACTCCTATCGGCAGCAGCAGC
AGCTCCTACAGAGGCCGAGTCACCTACAGC
CCCCGTCCTACGGGCCGGTCCTCTCGCCCA
TGAACAAGGTGCACGGGGGCATGAACAAGC
TGCCCTCCGTCAACCAGCTGGTGGGCCAGC
CTCCCCCGCACAGTTCGGCAGCTACACCCA
ACCTGGGGCCCGTGGGCCCCGGGATGCTCA
ACAACCATGGCCACGCAGTGCCAGCCAACG
GCGAGATGAGCAGCAGCCACAGCGCCCAGT
CCATGGTCTCGGGGTCCCACTGCACTCCGC
CACCCCCCTACCACGCCGACCCCAGCCTCG
TCAGTTTTTTAACAGGATTGGGGTGTCCAA
ACTGCATCGAGTATTTCACCTCCCAAGGGT
TACAGAGCATTTACCACCTGCAGAACCTGA
CCATTGAGGACCTGGGGGCCCTGAAGATCC
CCGAGCAGTACCGCATGACCATCTGGCGGG
GCCTGCAGGACCTGAAGCAGGGCCACGACT
ACAGCACCGCGCAGCAGCTGCTCCGCTCTA
GCAACGCGGCCACCATCTCCATCGGCGGCT
CAGGGGAACTGCAGCGCCAGCGGGTCATGG
AGGCCGTGCACTTCCGCGTGCGCCACACCA
TCACCATCCCCAACCGCGGCGGCCCAGGCG
GCGGCCCTGACGAGTGGGCGGACTTCGGCT
TCGACCTGCCCGACTGCAAGGCCCGCAAGC
AGCCCATCAAGGAGGAGTTCACGGAGGCCG
AGATCCACTGAGGGCCTCGCCTGGCTGCAG
CCTGCGCCACCGCCCAGAGACCCAAGCTGC
CTCCCCTCTCCTT

Final protein sequence
MAPVIPSNTDYPGPHHFEVTFQQSSTAKSA
TWTYSPLLKKLYCQIAKTCPIQIKVSTPPP
PGTAIRAMPVYKKAEHVTDVVKRCPNHELG
RDFNEGQSAPASHLIRVEGNNLSQYVDDPV
TGRQSVVVPYEPPQVGTEFTTILYNFMCNS
SCVGGMNRRPILIIITLEMRDGQVLGRRSF
EGRICACPGRDRKADEDHYREAENLYFQSH
HHHHHDYKDDDDK

Tags and additions: ENLYFQ*SHHHHHHDYKDDDDK, TEV-cleavable (*) C-terminal hexahistidine and FLAG tag.

Host: BL21(DE3)-R3-pRARE2

Growth medium, induction protocol: A glycerol stock was used to inoculate a 10ml starter culture containing LB media with 50µg/ml Kanamycin and 34 µg/ml chloramphenicol. The starter culture was grown overnight at 37°C with shaking at 200 rpm. The following morning, two flasks containing 1 L LB/kanamycin/chloramphenicol were each inoculated with 5 ml of the starter culture. Cultures were incubated at 37°C with shaking at 180 rpm until an OD₆₀₀nm ≥ 0.6 was reached. The flasks were then cooled down to 18°C. Protein expression was induced by addition of IPTG to a final concentration of 0.2mM and expression carried out overnight. Cells were harvested by centrifugation at 6000 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 30ml binding buffer (50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole), transferred to 50 ml tubes, and stored at -20°C.

Extraction buffer, extraction method: The frozen cells were thawed and 0.5mM TCEP, 1mM PMSF added to the cell suspension. The cells were lysed by ultrasonication over 12 min with the sonicator pulsing ON for 4 sec and OFF for 8 sec. The cell lysate was spun down by centrifugation at 20000 rpm at 4°C for 1 h. The supernatant was recovered for purification.

Column 1: Anion-exchange for Nucleic acid removal with DEAE cellulose (DE52, Whatmann)
10 g of resin was suspended in 100 ml 2.5 M NaCl, and then applied onto a 2.5 x 20 cm column. The resin was then equilibrated with 100 ml binding buffer prior to loading the sample.

Column 1 Buffers: Binding buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole Wash buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 30mM Imidazole

Column 1 Procedure: The supernatant was first applied onto the column by gravity flow, which was followed by a wash with 100 ml wash buffer. The column flow-through and wash was directly applied onto the following Ni-sepharose column.

Column 2: Ni-Affinity Chromatography
5ml of 50 % Ni-sepharose slurry (Amersham) was applied onto a 1.5 x 10 cm column. The column was first washed with deionised distilled H₂O, and then equilibrated with binding buffer.

Column 2 Buffers:
Binding buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 5mM Imidazole
Wash buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 30mM Imidazole
Elution buffer: 50mM HEPES, pH 7.5; 500mM NaCl; 5% Glycerol; 50 to 250mM Imidazole

Column 2 Procedure: The supernatant was applied by gravity flow onto the Ni-sepharose column. The column was washed with wash buffer to remove nonspecifically binding proteins. The bound target protein was eluted by applying a step gradient of imidazole (5 ml fractions of elution buffer supplemented with 50mM, 100mM, 150mM and 250mM imidazole). The protein content of collected fractions was visualized using SDS-PAGE and fractions containing sufficient TP73A (relative to contaminating proteins) were pooled. 10mM DTT was added to the pooled fractions for overnight storage at 4°C.

Enzymatic treatment: TEV protease cleavage. Pooled fractions were treated with TEV protease overnight at 4°C.

Column 3: Size Exclusion Chromatography - S200 HiLoad 16/60 Superdex run on ÄKTA-Express.

Column 3 Buffers: Gel Filtration buffer: 300mM NaCl, 50mM HEPES pH 7.5, 0.5mM TCEP

Column 3 Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. Eluted protein from the Ni-sepharose column was cleaved with TEV protease and concentrated to 1ml using an Amicon Ultra-15 filter with a 10kDa cut-off. The concentrated protein was directly applied onto the equilibrated S200 16/60 column, and run at a flow-rate of 1 ml/min. Fractions were visualized using SDS-PAGE and those containing TP73A were pooled. After concentration, the protein was divided into 100µl aliquots and stored at -80°C.

Protein concentration: The protein was concentrated to a final concentration of 19mg/ml (measured by OD₂₈₀ based on extinction coefficient 18910) in an Amicon Ultra-15 filter with a 10 kDa cut-off.

Mass spectrometry characterization: The purified native protein was homogeneous and had an experimental mass after tag cleavage of 23184.05 consistent with loss of the N-terminal initiator methionine (calculated MW of this species = 23313.6). Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser.

Crystallisation: Protein at 19mg/ml was buffered in 50mM HEPES, pH 7.5, 300mM NaCl, 0.5 mM TCEP. Crystals were grown at 20°C in 150 nl sitting drops mixing 50 nl protein solution with 100 nl of a reservoir solution containing 1.2M sodium potassium tartrate, 0.25% PEG MME 5000 and 0.1M Tris pH 9. On mounting crystals were cryo-protected with an additional 20% glycerol.

Data collection: Resolution: 1.8 Å; X-ray source: Diamond I02
Crystals of TP73A diffracted to a resolution of 1.8 Å (scaled resolution). A full dataset was collected at 100 K on Diamond Light Source beamline I02. Crystals belonged to the cubic space group P4₃32 with unit-cell parameters a=b=c= 110.32 Å. Only one molecule was present in the asymmetric unit. Data were indexed and integrated using iMOSFLM and scaled using SCALA. Phases were found using molecular replacement in PHASER. CHAINSAW was used to optimize the PDB entry 20CJ for use as a search model. The structure was refined and modified using alternate rounds of REFMAC5 and COOT, and the final model validated using the JCSG Quality Control Server.

References

Danilova, N., Sakamoto, K.M., and Lin, S. (2008). p53 family in development. Mech Dev 125, 919-931. PubMed 18835440
De Feo, E., Persiani, R., La Greca, A., Amore, R., Arzani, D., Rausei, S., D'Ugo, D., Magistrelli, P., van Duijn, C.M., Ricciardi, G., et al. (2009). A case-control study on the effect of p53 and p73 gene polymorphisms on gastric cancer risk and progression. Mutat Res 675, 60-65. PubMed 19386249
Li, F., Sturgis, E.M., Zafereo, M.E., Liu, Z., Wang, L.E., Wei, Q., and Li, G. (2009). p73 G4C14-to-A4T14 polymorphism and risk of second primary malignancy after index squamous cell carcinoma of head and neck. Int J Cancer 125, 2660-2665. PubMed 19585505
Loof, J., Pfeifer, D., Adell, G., and Sun, X.F. (2009). Significance of an exon 2 G4C14-to-A4T14 polymorphism in the p73 gene on survival in rectal cancer patients with or without preoperative radiotherapy. Radiother Oncol 92, 215-220. PubMed 19581013
Murray-Zmijewski, F., Lane, D.P., and Bourdon, J.C. (2006). p53/p63/p73 isoforms: an orchestra of isoforms to harmonise cell differentiation and response to stress. Cell Death Differ 13, 962-972. PubMed 16601753
Rosenbluth, J.M., and Pietenpol, J.A. (2008). The jury is in: p73 is a tumor suppressor after all. Genes Dev 22, 2591-2595. PubMed 18832062
Tomasini, R., Tsuchihara, K., Tsuda, C., Lau, S.K., Wilhelm, M., Ruffini, A., Tsao, M.S., Iovanna, J.L., Jurisicova, A., Melino, G., et al. (2009). TAp73 regulates the spindle assembly checkpoint by modulating BubR1 activity. Proc Natl Acad Sci U S A 106, 797-802. PubMed 19139399
Tournas, A., Mfuna, L., Bosse, Y., Filali-Mouhim, A., Grenier, J.P., and Desrosiers, M. A pooling-based genome-wide association study implicates the p73 gene in chronic rhinosinusitis. J Otolaryngol Head Neck Surg 39, 188-195. PubMed 20211107
Wilhelm, M.T., Rufini, A., Wetzel, M.K., Tsuchihara, K., Inoue, S., Tomasini, R., Itie-Youten, A., Wakeham, A., Arsenian-Henriksson, M., Melino, G., et al. Isoform-specific p73 knockout mice reveal a novel role for delta Np73 in the DNA damage response pathway. Genes Dev 24, 549-560. PubMed 20194434
Yang, A., and McKeon, F. (2000). P63 and P73: P53 mimics, menaces and more. Nat Rev Mol Cell Biol 1, 199-207. PubMed 11252895
Yang, A., Walker, N., Bronson, R., Kaghad, M., Oosterwegel, M., Bonnin, J., Vagner, C., Bonnet, H., Dikkes, P., Sharpe, A., et al. (2000). p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature 404, 99-103. PubMed 10716451
Zawacka-Pankau, J., Kostecka, A., Sznarkowska, A., Hedstrom, E., and Kawiak, A. p73 tumor suppressor protein: a close relative of p53 not only in structure but also in anti-cancer approach? Cell Cycle 9, 720-728. PubMed 20160513