Human haspin in complex with an Imidazo-pyridazine ligand

Target ID:

GSG2A

Aliases:

GSG2HASPIN

Deposition Date:

Thursday 30th October 2008

Families:

Protein Kinase

Related Diseases:

Chromatin and epigeneticsSignalling

Structure type:

protein-ligand

Download Coordinates:

3F2N

Authors:

Filippakopoulos, P., Eswaran, J., Keates, T., Burgess-Brown, N., Fedorov, O. Yue, W.W., Murray, J.W., Pike, A.C.W., Von Delft, F. Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C. Knapp, S.

Site:

Oxford

ICM Datapack:

ICM Datapack

3F2N

tabs

Structure Details

Haspin (haploid germ cell–specific nuclear protein kinase) is a serine/threonine kinase that tightly associates with chromosomes during mitosis. Haspin-like proteins are identified in several major eukaryotic phyla including yeasts, plants, flies, fish, and mammals. Kinases of this family share only very low sequence homology with other protein kinases, contain several large insertions and lack a subset of residues that are almost invariant in known kinases [1]. Phosphorylation of Haspin itself and its specific substrate histone H3 threonine-3 (H3T3ph) occurs during mitosis. Haspin has been shown to be vital for the maintenance of chromosome cohesion. Depletion of Haspin leads to a loss of cohesin association, premature chromatid separation, activation of the spindle assembly checkpoint, and a block in mitosis in a pro-metaphase-like state [2]. Although no direct link to cancer has been established so far, Haspin has been suggested as a target for drug development as it has been shown to activate the well established oncology target Aurora 2 [3].

Here we present the structure of the Haspin kinase domain refined at 1.8 Å resolution in complex with an imidazo[1.2-b]pyridazine inhibitor. We have also determined the structures of the Haspin kinase domain in complex with the ATP mimetic inhibitor 5-Iodotubercidin (4-Amino-5-iodo-7-(b-D-ribofuranosyl) pyrrolo[2,3-d]pyrimidine) (PDB ID 2VUW), with a pyrazolo[1.5a]pyrimidine inhibitor (PDB ID 3E7V) and with an isoquinoline inhibitor (PDB ID 3FMD). In order to get insight into ATP recognition we also determined the structure of Haspin in complex with ATP/Mg (PDB ID 3DLZ).

5-Iodotubercidin (4-amino-5-iodo-7-(D-ribofuranosyl) pyrrolo[2,3]pyrimidine)

(2VUW)

pyrazolo[1.5-a]pyrimidine

(3E7V) imidazo[1.2-b]pyridazine

(3F2N) isoquinoline

(3FMD)

Materials and Methods

Entry Clone Source: Synthetic

Entry Clone Accession: n/a

SGC Construct ID: GSG2A-c009

GenBank GI number: gi|56790919

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Tags and additions: Tag sequence: mhhhhhhssgvdlgtenlyfq*s(m) TEV-cleavable (*) N-terminal his6 tag.

Final protein sequence:
mhhhhhhssgvdlgtenlyfq*sMGECSQ
KGPVPFSHCLPTEKLQRCEKIGEGVFGEV
FQTIADHTPVAIKIIAIEGPDLVNGSHQK
TFEEILPEIIISKELSLLSGEVCNRTEGF
IGLNSVHCVQGSYPPLLLKAWDHYNSTKG
SANDRPDFFKDDQLFIVLEFEFGGIDLEQ
MRTKLSSLATAKSILHQLTASLAVAEASL
RFEHRDLHWGNVLLKKTSLKKLHYTLNGK
SSTIPSCGLQVSIIDYTLSRLERDGIVVF
CDVSMDEDLFTGDGDYQFDIYRLMKKENN
NRWGEYHPYSNVLWLHYLTDKMLKQMTFK
TKCNTPAMKQIKRKIQEFHRTMLNFSSAT
DLLCQHSLFK

Host: BL21(DE3)-R3-pRARE2 (previously known as Rosetta)

Growth medium, induction protocol: 1ml from a 10 ml overnight culture containing 50 µg/ml kanamycin and 35 µg/ml chloramphenicol was used to inoculate 1 liter of LB media containing the same concentration of antibiotics. Cultures were grown at 37°C until the OD₆₀₀ reached ~0.3. After that the temperature was adjusted to 20°C. Expression was induced over night using 1mM IPTG at an OD600 of 0.8. The cells were collected by centrifugation and the pellet resuspended in binding buffer and frozen. Binding buffer: 50mM HEPES pH 7.5; 500 mM NaCl; 5% glycerol, 10 mM imidazole.

Extraction method: Cell pellets were lysed by C5 high pressure homogenizer (Avestin). The lysate was centrifuged at 21,000 rpm for 60 minutes and the supernatant collected for purification.

Column 1: Ni-affinity chromatography.

Buffers: Binding buffer: Binding buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 5% Glycero, 10mM Imidazole. Wash buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 30 mM Imidazole, 5% glycerol. Elution buffer: 50 mM HEPES pH 7.5, 500 mM NaCl, 50 to 250 mM imidazole, 5% Glycerol.

Procedure: 5 ml of 50% Ni-NTA slurry (Qiagen) was applied to a 1.5 x 10 cm gravity column. The column was equilibrated with 50 ml binding buffer. The lysate was applied to the column which was subsequently washed with 50 ml wash buffer 1 and 2. The protein was eluted by gravity flow by applying 5 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150mM, 250 mM); fractions were collected until essentially all protein was eluted. The eluted protein was analyzed by SDS-PAGE. DTT was added to the protein sample to a final concentration of 5mM.

Column 2: Size exclusion chromatography (Superdex S75, 60 x 1cm)

SEC-Buffers: 50 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM DTT.

Procedure: The fractions eluted of the Ni-affinity chromatography were concentrated to about 4 mls using Centricon concentrators (10kDa cut off). The concentrated protein was applied to a Superdex S75 column equilibrated in SEC buffer at a flow rate of 0.8 ml/min. Eluted fractions were 95% pure as judged by SDS-PAGE.

Protein concentration: Centricon with a 30 kDa cut off in SEC-buffer.

Mass Spec Characterisation: The mass of the protein was calculated to be 40655 Da and experimentally determined mass was 40663 Da for the His tag containing protein. The identity of the protein was reconfirmed to be correct by DNA sequencing both DNA strands of this expression construct.

Crystallization: All crystallizations were carried out using sitting drop vapour diffusion at 4°C. Crystallization of protein was performed by matrix-seeding with crushed native crystals into 100nl drops composed of equal volumes of native protein (8mg/ml) with the ligand, ((S)-2-(3-(3-aminophenyl)imidazo[1,2-b]pyridazin-6-ylamino)-3-methylbutan-1-ol) (1mM) and reservoir solution containing: 0.10M Na/KPO₄, 20.0% PEG 3350 and 10.0% EtGly. Seeding crystals obtained from 100nl drops composed of equal volume of protein (14mg/ml) and reservoir solution containing: 0.1M MMT pH 6.0; 60.0% MPD.

Data Collection: Crystals were cryo-protected using the well solution supplemented with 25% Ethylene Glycol and flash frozen in liquid nitrogen. Diffraction data were collected from a single crystal at the Swiss Light Source (PX10) on a single wavelength of 0.9789 Å. The structure was solved by molecular replacement and refined to 1.80 Å.

References

1. Higgins, J.M., Haspin-like proteins: a new family of evolutionarily conserved putative eukaryotic protein kinases. Protein Sci, 2001. 10(8): p. 1677-84.
2. Dai, J., et al., The kinase haspin is required for mitotic histone H3 Thr 3 phosphorylation and normal metaphase chromosome alignment. Genes Dev, 2005. 19(4): p. 472-88.
3. Rosasco-Nitcher, S.E., et al., Centromeric Aurora-B activation requires TD-60, microtubules, and substrate priming phosphorylation. Science, 2008. 319(5862): p. 469-72.