poly (ADP-ribose) polymerase 2, catalytic fragment in complex with ABT888

Target ID:

PARP2A

Aliases:

ADPRT2ADPRTL2ADPRTL3pADPRT-2PARP-2PARP2

Deposition Date:

Thursday 22nd October 2009

Families:

Poly ADP-ribose polymerase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3KJD

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, A.JOHANSSON, I.JOHANSSON, A.KALLAS, T.KOTENYOVA, A.KOTZSCH, P.KRAULIS, T.K.NIELSEN, M.MOCHE, P.NORDLUND, T.NYMAN, C.PERSSON, A.K.ROOS, M.I.SIPONEN, A.G.THORSELL, L.TRESAUGUES, S.VAN DEN BERG, J.WEIGELT, M.WELIN, M.WISNIEWSKA, H.SCHULER

Site:

Stockholm

3KJD

tabs

Structure Details

Poly(ADP-ribose) polymerases (PARPs) are enzymes that use NAD+ as a substrate to add poly(ADP)ribose (PAR) to other proteins or themselves, leading to a changed three-dimensional and electrostatic surface of the modified proteins (1). PAR is critically involved in genome organization, transcriptional regulation, and DNA repair (2). The human PARP family consists of 17 proteins that all contain a catalytic domain in the context of different domains conferring interactions with other proteins and subcellular localization (3). PARP-1 is triggered by DNA breaks and its activation initiates recruitment of the DNA repair machinery, and inhibiting this pathway is considered as a promising avenue for treatment of cancers in which mutations in the DNA damage response machinery are important (1). Several inhibitors have been developed that target the NAD+-binding site and inhibit the activity of PARP-1 and/or PARP-2.

PARP-2 (4) is the family member with greatest homology to PARP-1; PARP-2 and PARP-1 form functional heterodimers (5,6) but the two proteins may have overlapping functions (7). Here, the crystal structure of the catalytic domain of human PARP-2 is presented in complex with the NAD+-analogue inhibitor ABT-888 at 1.95Å resolution. ABT-888 has high (nanomolar) affinity towards PARP-1 and -2 (8). The structure consists of an N-terminal α-helical lobe and a C-terminal PARP domain similar to both PARP-1 and PARP-3. The PARP domain contains the PARP signature motif (9), a diphtheria toxin like fold (β-α-loop-β-α) on its inner surface, forming the NAD+ donor binding site located between the two subdomains. This structure may inform the rational design of inhibitors that are specific and selective within the PARP-1/ -2/ -3 clade.

Materials and Methods

PARP2 + ABT888

PDB:3KJD

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:E0050
Entry Clone Source:GeneCopoeia
SGC Clone Accession:PARP2A-s002, PARP2A-s003
Tag:N-terminal hexahistidine tag: MHHHHHHSSGVDLGTENLYFQSM
Host:Escherichia coli BL21(DE3) R3 pRARE

Construct

Prelude:
Sequence:MHHHHHHSSGVDLGTENLYFQSMDLRVQELIKLICNVQAMEEMMMEMKYNTKKAPLGKLTVAQIKAGYQSLKKIEDCIRAGQHGRALMEACNEFYTRIPHDFGLRTPPLIRTQKELSEKIQLLEALGDIEIAIKLVKTELQSPEHPLDQHYRNLHCALRPLDHESYEFKVISQYLQSTHAPTHSDYTMTLLDLFEVEKDGEKEAFREDLHNRMLLWHGSRMSNWVGILSHGLRIAHPEAPITGYMFGKGIYFADMSSKSANYCFASRLKNTGLLLLSEVALGQCNELLEANPKAEGLLQGKHSTKGLGKMAPSSAHFVTLNGSTVPLGPASDTGILNPDGYTLNYNEYIVYNPNQVRMRYLLKVQFNF
Vector:pNIC-Bsa4

Growth

Medium:Fresh overnight cultures of E. coli strain BL21(DE3) R3 pRARE cells (including 100 µg/ml kanamycin and 34 µg/ml chloramphenicol) transformed with PARP2 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 ~2. The culture was down-tempered to 18°C for 1 h. Expression of PARP2 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 (107g 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.
Antibiotics:
Procedure:

Purification

Procedure
Columns
IMAC: Ni-charged 5 ml HiTrap Chelating HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 200 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. The protein was concentrated using an Amicon Ultra-15 centrifugal filter device, 10,000 MWCO (Millipore) to 37.2 mg/ml in a volume of 0.6 ml. The expected mass of the protein was confirmed by mass spectrometry.

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
ABT888MassSpec:
Crystallization:Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.2 µl protein solution (30 mg/ml) including 4 mM ABT888 was mixed with 0.1 µl of well solution consisting of 25% PEG3350, 0.1M Tris-HCl pH 8.5, 0.25M NaCl. The plate was incubated at 4 ºC. Crystals grew in 2 weeks wherafter they were quickly transferred to a cryo solution consisting of 25% PEG3350, 0.1M Tris-HCl pH 8.5, 15% Glycerol, 0.3M NaC, 1mM ABT888l, and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 1.95 Å resolution was collected at DIAMOND beamline I04.
Data Processing:Data were indexed and integrated in space group P21 using XDS software. The structure was solved by molecular replacement using the structure of the catalytic domain of the previously determined human parp2 (pdb: 3KCZ) as model template. Molrep was used to solve the structure. The asymmetric unit contained two protein monomers. The space group was P21 with cell dimensions a = 58.13Å, b = 134.61Å, c = 58.31Å, β = 117.68°. Arp/warp was used for initial automatic model building, Refmac5 for refinement and Coot for manual model building. Data in the interval 41.0-1.95 Å resolution were used and refined to R = 18.16% and Rfree = 23.19%. Coordinates for the crystal structure were deposited in the Protein Data Bank, with accession code 3KJD.

References

Hassa PO and Hottiger MO. (2008) The diverse biological roles of mammalian PARPs, a small but powerful family of poly-ADP-ribose polymerases. Front. Biosci. 13, 3046-3082.
PubMed 17981777
Schreiber V et al. (2006) Poly(ADP-ribose): novel functions for an old molecule. Nature Rev. Mol. Cell Biol. 7, 517-528.
PubMed 16829982
Amé JC et al. (2004) The PARP superfamily. Bioessays 26, 882-893.
PubMed 15273990
Amé JC et al. (1999) PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J Biol Chem 274, 17860-17868.
PubMed 10364231
Schreiber V et al. (2002) Poly(ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1. J Biol Chem 277, 23028-23036.
PubMed 11948190
Bryant HE et al. (2009) PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination. EMBO J. 28, 2601-2615.
PubMed 19629035
Menissier de Murcia J et al. (2003) Functional interaction between PARP-1 and PARP-2 in chromosome stability and embryonic development in mouse. EMBO J. 22, 2255-2263.
PubMed 12727891
Penning TD et al. (2009) Discovery of the Poly(ADP-ribose) polymerase (PARP) inhibitor 2-[(R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide (ABT-888) for the treatment of cancer. J Med Chem 52, 514-523.
PubMed 19143569
Otto H et al. (2005) In silico characterization of the family of PARP-like poly(ADPribosyl) transferases (pARTs). BMC Genomics 6, 139.
PubMed 16202152