Human ABL2 in complex with Aurora Kinase Inhibitor VX-680

Target ID:

ABL2A

Aliases:

ABL2ABLLARGFLJ22224FLJ31718FLJ41441

Deposition Date:

Friday 2nd July 2010

Families:

Protein Kinase

Related Diseases:

CancerSignallingNeurobiology

Structure type:

protein-ligand

Download Coordinates:

2XYN

Authors:

Salah, E.; Ugochukwu, E.; Elkins, J.; Barr, A.; Shrestha, B.; Savitsky, P.; Mahajan, P.; Muniz, J.; Yue, W. W.; Chaikuad, A.; von Delft, F.; Bountra, C.; Arrowsmith, C. H.; Weigelt, J.; Edwards, A.; Knapp, S.

Site:

Oxford

2XYN

tabs

Structure Details

Abl2 (also known as ARG (ABL Related Gene)) is a member of the Abelson family of non-receptor tyrosine kinase. ABL2 shares a high degree of sequence conservation with cABL and has similar domain organization comprising an N-terminal SH3?SH2 kinase domain unit followed by a C-terminal domain containing docking sites for SH3 domains, F-actin and microtubules. Through its interactions with the cytoskeleton ABL2 plays a role in murine neurulation and it is required for adhesion-dependent neurite branching and synapse/dendrite stability. ABL2 has also a role in fibroblastic- and epithelial cell adhesion and migration. ABL2 shares a number of cellular regulatory functions with cABL. Targeted disruption of the cAbl gene in mice resulted in pleiotropic phenotypes including runtedness, high perinatal lethality, susceptibility to infections, and immune deficiencies. In contrast, homozygous Abl2 knockout mice are healthy and exhibit no immune-deficient phenotypes. However, evidence for overlapping roles of cAbl and Abl2 was obtained by analyzing mice deficient for both genes, which revealed that both proteins are required for normal mouse development. Abl/Abl2-/- mice die around embryonic day 10.5, and the embryos display bleeding in the pericardial sac, exhibit reduced proliferation and cytokine production in response to TCR stimulation and showed a significantly impaired antibody production.

Like ABL1, ABL2 is involved in human neoplastic diseases. Abl is up- or down-regulated in several solid tumors and oncogenic gene translocations involving the ETV6 gene have been described in human acute leukemia. Here we describe the structure of the kinase domain of ABL2 at 2.05Å resolution in complex with the oncology drug gleevec. In addition we crystallized ABL2 in complex with a type I inhibitor (1-acyl- 1H- [1,2,4] triazole- 3,5- diamine) and determined the structure at 1.65Å resolution. This inhibitor has been reported as a potent cdk1 inhibitor with significant antitumor activity. Interestingly, gleevec (imatinib) interacts not only with the ATP binding site but was found to bind also to the regulatory myristilation site. The structure of this complex may therefore serve as a tool for the development of allosteric ABL inhibitors. Here we report the structure of ABL2 in complex with VX680 a potent type II inhibitor that has been developed as a specific Aurora kinase inhibitor.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE:5173213

SGC Construct ID: ABL2A-c055

GenBank GI number: gi|6382060

Vector: pFB-LIC-Bse. Details [ PDF ]; Sequence [ FASTA ] or [ GenBank ]

Amplified construct sequence:
TACTTCCAATCCATGGACAAATGGGA
AATGGAGCGAACAGATATTACCATGA
AGCACAAACTTGGGGGCGGTCAGTAT
GGAGAGGTTTACGTTGGCGTCTGGAA
GAAATACAGCCTTACAGTTGCTGTGA
AAACATTGAAGGAAGATACCATGGAG
GTAGAAGAATTCCTGAAAGAAGCTGC
AGTAATGAAGGAAATCAAGCATCCTA
ATCTGGTACAACTTTTAGGTGTGTGT
ACTTTGGAGCCACCATTTTACATTGT
GACTGAATACATGCCATACGGGAATT
TGCTGGATTACCTCCGAGAATGCAAC
CGAGAAGAGGTGACTGCAGTTGTGCT
GCTCTACATGGCCACTCAGATTTCTT
CTGCAATGGAGTACTTAGAGAAGAAG
AATTTCATCCATAGAGATCTTGCAGC
TCGTAACTGCCTAGTGGGAGAAAACC
ATGTGGTAAAAGTGGCTGACTTTGGC
TTAAGTAGATTGATGACTGGAGACAC
TTATACTGCTCATGCTGGAGCCAAAT
TTCCTATTAAGTGGACAGCACCAGAG
AGTCTTGCCTACAATACCTTCTCAAT
TAAATCTGACGTCTGGGCTTTTGGGG
TATTGTTGTGGGAAATTGCTACCTAT
GGAATGTCACCATATCCAGGTATTGA
CCTGTCTCAGGTCTATGACCTACTAG
AAAAAGGATATCGAATGGAACAGCCT
GAGGGATGCCCCCCTAAGGTTTATGA
ACTTATGAGAGCATGCTGGAAGTGGA
GCCCTGCCGATAGGCCCTCTTTTGCT
GAAACACACCAAGCTTTTGAAACCAT
GTTCCATGACTCTTGACAGTAAAGGT
GGATA

Final protein sequence (Tag sequence in lowercase):
mghhhhhhssgvdlgtenlyfq^sMD
KWEMERTDITMKHKLGGGQYGEVYVG
VWKKYSLTVAVKTLKEDTMEVEEFLK
EAAVMKEIKHPNLVQLLGVCTLEPPF
YIVTEYMPYGNLLDYLRECNREEVTA
VVLLYMATQISSAMEYLEKKNFIHRD
LAARNCLVGENHVVKVADFGLSRLMT
GDTYTAHAGAKFPIKWTAPESLAYNT
FSIKSDVWAFGVLLWEIATYGMSPYP
GIDLSQVYDLLEKGYRMEQPEGCPPK
VYELMRACWKWSPADRPSFAETHQAF
ETMFHDS

^ TEV cleavage site

Tags and additions: Cleavable N-terminal His6 tag.

Host: Baculo Virus infected Insect cells (High5 cells).

Growth medium, induction protocol: High five cells grown in Insect Express Medium. Cells were infected at a density of 2x10⁶/ml with recombinant baculovirus (virus stock P2; 1ml of virus stock/100ml of cell culture). Cells were shaken at 120rpm at 27°C in the innova shaker. After 48 hours post-infection the cultures were collected and centrifuged for 10min at 2000rpm. The cell pellet was resuspended in cold PBS and centrifugation was repeated.
Binding buffer: 50 mM HEPES pH 7.5; 500 mM NaCl; 5 mM Imidazole, 5% Glycerol; 0.5 mM TCEP.
Extraction buffer, extraction method: Frozen pellets were thawed and cells lysed using a high pressure cell disrupter. PEI (polyethleneimine) was added to the lysate to a final concentration of 0.15% and the lysate was centrifuged at 17,000rpm for 30 minutes and the supernatant collected for purification.

Column 1: Ni-affinity. Ni-NTA (Qiagen), 5ml of 50% slurry in 1.5 x 10cm column, washed with binding buffer.

Column 1 Buffer:
Binding buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5mM TCEP.
Wash buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5mM TCEP; 20 mM Imidazole.
Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol; 0.5mM TCEP; 300 mM Imidazole.

Column 1 Procedure: The cleared lysate was loaded by gravity flow on the Ni-NTA column. The column was then washed with 100ml binding buffer and 100ml wash buffer at gravity flow. The protein was eluted by gravity flow by applying 5ml of elution buffer.

Column 2: Size Exclusion Chromatography. Superdex S200 16/60 HiLoad

SEC-Buffers:
For the gleevec complex (3GVU): 10 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 0.5 mM TCEP.
For the triazole-carbothioamide complex (3HMI): 25 mM HEPES, pH 7.5; 300 mM NaCl; 0.5 mM TCEP.

Column 2 Procedure: The protein was concentrated and applied to an S200 16/60 HiLoad gel fitration column equilibrated in SEC-Buffer using ÄKTA express system.

Protein concentration: Protein was concentrated to 4.0mg/ml (gleevec complex; 3GVU) and 8.0mg/ml (triazole-carbothioamide complex; 3HMI) and 10mg/mL for the VX-680 complex, using an Amicon 10kDa cut-off concentrator.

Mass spectrometry characterization: For the Gleevec complex (3GVU), the mass of the protein was calculated to be 33502Da and experimentally determined mass was 33414Da for the His tag containing protein. It is therefore likely that the difference in mass is due to removal of the initial Met and Acetylation. For the triazole-carbothioamide complex (3HMI), the mass of the protein was calculated to be 30980 Da after His tag cleavage and experimentally determined mass was 30980 Da.

Crystallisation: The ABL2:imatinib complex (3GVU) was crystallised at 4°C in 200nl drops from a 1:1 ratio of ABL2:imatinib (4mg/ml protein containing 1 mM imatinib) and reservoir solution (20% PEG3350, 0.1 M citrate pH 5.5). The ABL2:JNJ-7706621 complex (3HMI) was crystallised at 4°C in 200nl drops from a 3:1 ratio of ABL2:JNJ-7706621 (8 mg/ml protein containing 1 mM JNJ-7706621 (5-Amino-3-((4-(aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-1H-1,2,4-triazole-1-carbothioamide, Calbiochem product #217714) and reservoir solution (0.1 M lithium sulfate, 0.05 M di-sodium hydrogen phosphate, 0.05 M citric acid, 19% (v/v) PEG1000). The ABL2:VX-680 complex (2XYN) was crystallised at 4°C in 200nl drops from a 2:1 ratio of ABL2:JNJ-7706621 (10 mg/ml protein containing 1 mM VX-680 (GSK487830B)) and reservoir solution (0.8M Sodium succinate).

Data Collection: Crystals were cryo-protected using 25% ethylene glycol for the gleevec complex and 20% PEG300 for the triazole-carbothioamide complex, respectively..
X-ray source: Diffraction data were collected at the SLS-X10SA (gleevec complex; 3GVU) and at the Diamond beam line I03 (triazole-carbothioamide complex (3HMI) and VX-680 (2XYN).
Phasing: 2.05Å (gleevec complex; 3GVU), 1.65Å (triazole-carbothioamide complex; 3HMI), 2.81Å (VX-680 complex).

References

De Keersmaecker, K., and Cools, J. (2006). Chronic myeloproliferative disorders: a tyrosine kinase tale. Leukemia 20, 200-205.
Griesinger, F., Janke, A., Podleschny, M., and Bohlander, S.K. (2002). Identification of an ETV6-ABL2 fusion transcript in combination with an ETV6 point mutation in a T-cell acute lymphoblastic leukaemia cell line. British journal of haematology 119, 454-458.
Gu, J.J., Ryu, J.R., and Pendergast, A.M. (2009). Abl tyrosine kinases in T-cell signaling. Immunological reviews 228, 170-183.
Iijima, Y., Ito, T., Oikawa, T., Eguchi, M., Eguchi-Ishimae, M., Kamada, N., Kishi, K., Asano, S., Sakaki, Y., and Sato, Y. (2000). A new ETV6/TEL partner gene, ARG (ABL-related gene or ABL2), identified in an AML-M3 cell line with a t(1;12)(q25;p13) translocation. Blood 95, 2126-2131.
Yagasaki, F., Wakao, D., Yokoyama, Y., Uchida, Y., Murohashi, I., Kayano, H., Taniwaki, M., Matsuda, A., and Bessho, M. (2001). Fusion of ETV6 to fibroblast growth factor receptor 3 in peripheral T-cell lymphoma with a t(4;12)(p16;p13) chromosomal translocation. Cancer research 61, 8371-8374.
Yuan, B.Z., Jefferson, A.M., Popescu, N.C., and Reynolds, S.H. (2004). Aberrant gene expression in human non small cell lung carcinoma cells exposed to demethylating agent 5-aza-2'-deoxycytidine. Neoplasia (New York, NY) 6, 412-419.