Human Tankyrase 1 - catalytic PARP-domain

Target ID:

TNKS1A

Aliases:

PARP-5aPARP5APARPLTIN1TINF1TNKSTNKS1

Deposition Date:

Friday 28th September 2007

Families:

Poly ADP-ribose polymerase

Related Diseases:

Cancer

Structure type:

apo

Download Coordinates:

2RF5

Authors:

LEHTIO, L., KARLBERG, T., ARROWSMITH, C., BERGLUND, H., BUSAM, R., COLLINS, R., DAHLGREN, L.G., EDWARDS, A., FLODIN, S., FLORES, A., GRASLUND, S., HAMMARSTROM, M., HERMAN, M.D., HOLMBERG-SCHIAVONE, L., JOHANSSON, I., KALLAS, A., KOTENYOVA, T., MOCHE, M., NORDLUND, P., NYMAN, T., PERSSON, C., SAGEMARK, J., SUNDSTROM, M., THORSELL, A.G., TRESAUGUES, L., VAN DEN BERG, S., WELIN, M., WEIGELT, J.

Site:

Stockholm

ICM Datapack:

ICM Datapack

2RF5

tabs

Structure Details

Tankyrase (TRF1-interacting ankyrin related ADP-ribose polymerase) was first identified in 1998 (1) and found to interact with TRF1 - a component of the shelterin complex, which binds to, and protects, telomeric DNA. After its discovery, tankyrase has been shown to interact with various proteins linking it to telomere homeostasis and mitosis (2).

Tankyrase belongs to the 17 membered family of PARP-enzymes (3), that use NAD+ as a substrate to add poly-ADP-ribose to other proteins or themselves. Poly ADP-ribosylation is a covalent modification that often modulates the affinity of the target molecule towards its binding partners (3). PARP-1 is an established drug target for cancer and several PARP-1 inhibitors are currently in clinical trials (4). Tankyrases are modular proteins that, in addition to the catalytic PARP domain, also contain: HPS-, ANK-, and SAM-domains. The two human tankyrases, TNKS1 and 2 are very homologous in sequence, but TNKS2 lacks the N-terminal HSP-domain.

In telomere length control TNKS1 binds and poly-ADP ribosylates the shelterin component TRF1. This modification disrupts the shelterin complex and exposes telomeres to telomerase, facilitating elongation of the telomeric DNA. Telomerase activity is increased in several cancer cells and inhibition of telomerase activity provides an opportunity to develop new cancer therapies (5). Tankyrase inhibition has been demonstrated as an attractive mechanism to augment the effect of direct telomerase inhibition (6). The present structure of the catalytic PARP domain of TNKS1 will enable structure based design of specific TNKS1 inhibitors, in particular, and will add to the structural knowledge that provides a framework for development of selective PARP inhibitors in general (7).

Materials and Methods

TNKS1

PDB:2RF5

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:BC098394
Entry Clone Source:Mammalian Gene Collection
SGC Clone Accession:
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*s(m)
Host:E.coli Rosetta2(DE3) (Novagen)

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfq*sMQGTNPYLTFHCVNQGTILLDLAPEDKEYQSVEEEMQSTIREHRDGGNAGGIFNRYNVIRIQKVVNKKLRERFCHRQKEVSEENHNHHNERMLFHGSPFINAIIHKGFDERHAYIGGMFGAGIYFAENSSKSNQYVYGIGGGTGCPTHKDRSCYICHRQMLFCRVTLGKSFLQFSTIKMAHAPPGHHSVIGRPSVNGLAYAEYVIYRGEQAYPEYLITYQIMKPEAPSQTATAAEQ
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were streaked onto LB-agar plates. 5-10 colonies were used to inoculate 20 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol. The cells were grown at 30 ºC overnight. The overnight culture (20 ml) was used to inoculate 1.5 l TB supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 200 µl PPG P2,000 81380 anti-foam solution (Fluka). The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 ºC until OD600 reached ~2. The culture was down-tempered to 18 ºC over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (5,500 x g, 10 min, 4 ºC). The resulting cell pellet (38.2 g wet cell weight) was resuspended in lysis buffer (2 ml/g cell pellet), supplemented with one tablet of Complete EDTA-free protease inhibitor (Roche Applied Science). The cell suspension was stored at -80 ºC.

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 as a two step process on an ÄKTAxpress system (GE Healthcare). Prior to purification, columns were equilibrated with IMAC wash1 buffer and gel filtration buffer, respectively. The filtered lysate was loaded onto the Ni-charged HiTrap Chelating column and 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. Fractions containing the target protein were pooled and fresh TCEP was added to a final concentration of 2 mM. The protein was subsequently concentrated using a Amicon Ultra-15 centrifugal filter device, 10,000 NMWL (Millipore) to 22.8 mg/ml in a volume of 0.28 ml. The identity of the protein was confirmed by mass spectrometry.

Extraction

Procedure
The cell suspension was quickly thawed in water and 2500 U Benzonase (Merck) was added. 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 (49,100 x g, 20 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl protein solution (22.8 mg/ml) was mixed with 0.1 µl of well solution consisting of 0.18 M Mg-formate pH 5.9, 3% 1,6-hexanediol, 18% PEG-3350. The plate was incubated at 20 ºC and a rod shaped crystals appeared in three days. The crystal was quickly transferred to a cryo solution consisting of well solution complemented with 0.3 M NaCl and 20% glycerol and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Data to 2.3 Å resolution was collected at BESSY beamline 14.1. As data contained intense ice rings a lower resolution dataset from identically grown crystal was also collected. After removal of ice rings two datasets were scaled together. Data were processed with XDS in space group I212121 (a=79.95 Å, b=81.24 Å and c=82.72 Å).
Data Processing:The structure was solved by molecular replacement with MOLREP using the PARP12 (PDB code 2PQF) model with truncated side chains as a search model. Model building was done with COOT and refinement with REFMAC5. TLS refinement with 4 TLS groups was used in the refinement process. At the end of the refinement the R values were: R= 19.3% and Rfree= 25.0%.

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.
Hsiao, S.J. and Smith, S. (2007) Tankyrase function at telomeres, spindle poles, and beyond. Biochimie. In press.
Schreiber, V., Dantzer, F., Amé, J.C. and de Murcia, G. (2006) Poly(ADP-ribose): novel functions for an old molecule. Nat. Rev. Mol. Cell Biol., 7, 517-528.
Ratnam, K. and Low, J.A. (2007) Current Development of Clinical Inhibitors of Poly(ADP-Ribose) Polymerase in Oncology. Clin. Cancer. Res., 13, 1383-1388.
Zimmermann, S. and Martens, U.M. (2007) Telomeres and telomerase as targets for cancer therapy. Cell. Mol. Life Sci., 64, 906-921.
Seimiya, H. (2006) The telomeric PARP, tankyrases, as targets for cancer therapy. Br. J. Cancer, 94, 341-345.
Lehtiö L, Collins R, van den Berg S, Johansson A, Dahlgren L-G, Hammarström, M, Helleday T, Holmberg-Schiavone, L, Karlberg T and Weigelt J. Zinc Binding Catalytic Domain of Human Tankyrase 1. JMB 2008 May 23;379(1):136-45.