Human tankyrase 2 - catalytic PARP domain in complex with 1-methyl-3-(trifluoromethyl)-5h-benzo[c][1,8]naphtyridine-6-one

Target ID:

TNKS2A

Aliases:

PARP-5bPARP-5cPARP5BPARP5CTANK2TNKLTNKS2

Deposition Date:

Thursday 8th April 2010

Families:

Poly ADP-ribose polymerase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3MHJ

Authors:

T.KARLBERG,P.SCHUTZ,C.H.ARROWSMITH,H.BERGLUND,C.BOUNTRA, R.COLLINS,A.M.EDWARDS,S.FLODIN,A.FLORES,S.GRASLUND, M.HAMMARSTROM,I.JOHANSSON,T.KOTENYOVA,N.MARKOVA, M.MOCHE,P.NORDLUND,T.NYMAN,C.PERSSON,M.I.SIPONEN,L.SVENSSON,A.G.THORSELL,L.TRESAUGUES,S.VAN DEN BERG,J.WEIGELT,M.WELIN,M.WISNIEWSKA,H.SCHULER

Site:

Stockholm

3MHJ

tabs

Structure Details

Tankyrases are poly-ADP-ribose transferases, owing to their C-terminal PARP-homology domain (1-5). Tankyrases are positive regulators of telomere elongation; they poly-ADP-ribosylate and displace TRF1 from telomeric repeats thereby allowing telomerase access to telomere ends (1,5). Tankyrases also mark the β-catenin destruction complex component axin for degradation, and tankyrase inhibition antagonizes the Wnt signal transduction pathway by stimulating β-catenin degradation (6). Inhibition of tankyrase activity imposes selective lethality on BRCA deficient cell lines (7). Thus, inhibition of tankyrase activity is a promising strategy for targeting cancer by diverse mechanisms.

Here we present the crystal structure of the human tankyrase-2 PARP domain in complex with an inhibitor 1-methyl-3-(trifluoromethyl)-5h-benzo[c][1,8]naphtyridine-6-one at 1.8 Å. The structure was obtained after soaking apo crystals (pdb entry 3KR7) with the compound. The complex structure shows binding of the ligand to the nicotinamide pocket as in other PARP ligand complexes, but additional specific contacts are of mainly non-polar nature. Interestingly, the D-loop takes a different conformation comparing with Tankyrase in complex with the potent inhibitor XAV939 (8).

Materials and Methods

TNKS2

PDB:3MHJ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NM_025235
Entry Clone Source:Origene
SGC Clone Accession:TNKS2A-s001
Tag:N-terminal hexahistidine tag: mhhhhhhssgvdlgtenlyfqsm
Host:Escherichia coli BL21(DE3) R3 pRARE

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsm*LNTSGSGTILIDLSPDDKEFQSVEEEMQSTVREHRDGGHAGGIFNRYNILKIQKVCNKKLWERYTHRRKEVSEENHNHANERMLFHGSPFVNAIIHKGFDERHAYIGGMFGAGIYFAENSSKSNQYVYGIGGGTGCPVHKDRSCYICHRQLLFCRVTLGKSFLQFSAMKMAHSPPGHHSVTGRPSVNGLALAEYVIYRGEQAYPEYLITYQIMRPEG
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Fresh overnight cultures of E. coli strain BL21(DE3) R3 pRARE cells (including 100 µg/ml kanamycin and 34 µg/ml chloramphenicol) transformed with TNKS2 expression construct were used to inoculate 4.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and anti-foam PPG P2000 in three 2-liter flasks. Cells were grown in a large scale expression system (Harbinger Biotechnology and Engineering) at 37°C until the OD600 reached ~1.6. The culture was down-tempered to 18°C for 1 h. Expression of TNKS2 was induced by adding 0.5 mM IPTG and growth continued over night at 18°C. Cells were harvested by centrifugation at 4400 x g for 10 min. The pellet (29g wet cell weight) was resuspended in 90ml lysis buffer supplemented with Complete EDTA-free Protease Inhibitor (Roche Biosciences) and benzonase Suspended cells were stored at -80°C until further use.

Purification

Procedure
Columns
IMAC: Ni-charged 1 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)

Procedure
Purification of the protein was performed on an ÄKTAxpress system (GE Healthcare). Prior to purification, IMAC column was equilibrated with IMAC wash1 buffer and gel filtration column with gel filtration buffer. The filtered lysate was loaded onto the IMAC column, and therafter washed with IMAC wash1 buffer followed by IMAC wash2 buffer. Bound protein was eluted from the IMAC column with IMAC elution buffer and automatically loaded onto the gel filtration column. Eluting fractions were analyzed by SDS-PAGE and target protein was pooled. Fresh TCEP was added to a final concentration of 2 mM. Purified protein was difficult to concentrate using an Amicon Ultra-15 centrifugal filter device (Millipore, 10,000 MWCO). Chymotrypsin (1:100 w/w) was added and the protein was concentrated to 13.7 mg/ml. Mass spectrometry, identified two chymotrypsin cleaved TNKS2 fragments.

Extraction

Procedure
The cell suspension was quickly thawed in water. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 3 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49000 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
1-methyl-3-(trifluoromethyl)-5h-benzo[c][1,8]naphtyridine-6-oneMassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (13.7 mg/ml) was mixed with 0.1 µl of well solution consisting of 17% PEG3350, 0.2M ammonium sulfate, 0.1M Tris-HCl pH 7.5. The plate was incubated at 4 ºC. Crystals grew in one week wherafter 1-methyl-3-(trifluoromethyl)-5h-benzo[c][1,8]naphtyridine-6-one was added and drops were incubated for one week. Crystals were quickly transferred to a cryo solution consisting of 19% PEG3350, 0.2M ammonium sulfate, 0.1M Tris-HCl pH 7.5, 23% Glycerol, 0.2M NaCl and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 1.8 Å resolution was collected at ESRF beamline ID-29 using wavelength 0.97908Å.
Data Processing:Data were indexed and integrated in space group C222₁ using XDS software. The structure was solved by molecular replacement using the human tankyrase-2 apo structure (pdb: 3kr7) as model template. Molrep was used to solve the structure. The asymmetric unit contained two protein monomers. The cell dimensions are a = 90.6Å, b = 97.7Å, c = 119.1Å. Refmac5 was used for refinement and Coot for model building. Data in the interval 45.0-1.8 Å resolution were used and refined to R = 17.30% and R_free = 20.28%. Coordinates for the crystal structure were deposited in the Protein Data Bank, with accession code 3mhj.

References

Smith S, Giriat I, Schmitt A and de Lange T (1998) Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282, 1484-1487. PubMed
Kaminker PG et al. (2001) TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression. J. Biol. Chem. 276, 35891-35899. PubMed
Amé JC et al. (2004) The PARP superfamily. Bioessays 26, 882-893. PubMed
Hsiao SJ and Smith S (2008) Tankyrase function at telomeres, spindle poles, and beyond. Biochimie 90, 83-92. PubMed
Smith S and de Lange T (1998) Tankyrase promotes telomere elongation in human cells. Curr. Biol. 10, 1299-1302. PubMed
Huang SM et al. (2009) Tankyrase inhibition stabilizes axin and antagonizes Wnt signaling. Nature 461, 614-620. PubMed
McCabe N et al. (2009) Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer. Oncogene 28, 1465-1470. PubMed
Karlberg T et al. (2010) Structural basis for the interaction between tankyrase-2 and a potent Wnt-signaling inhibitor. J. Med. Chem. 53, 5352-5355. PubMed