Human ACVR1 kinase domain in complex with the imidazo[1,2-b]pyridazine inhibitor K00135

Target ID:

ACVR1A

Aliases:

ACTRIACVR1ACVR1AACVRLK2ALK2FOPSKR1TSRI

Deposition Date:

Wednesday 29th February 2012

Families:

Nucleotide metabolismProtein Kinase

Related Diseases:

CancerSignalling

Structure type:

protein-ligand

Download Coordinates:

4DYM

Authors:

Chaikuad, A., Sanvitale, C., Cooper, C., Canning, P., Mahajan, P., Daga, N., Petrie, K., Alfano, I., Gileadi, O., Fedorov, O., Krojer, T., Filippakopoulos, P., Muniz, J.R.C., von Delft, F., Weigelt, J., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Bullock, A., Structural Genomics Consortium (SGC)

Site:

Oxford

4DYM

tabs

Structure Details

ACVR1 (also known as ALK2) belongs to the bone morphogenetic protein (BMP) receptor family of transmembrane serine/threonine kinases. Secreted BMPs, which are members of the TGF-beta superfamily involved in endochondral bone formation and embryogenesis, induce heteromeric assembly of type II and type I receptors. Five type II and seven type I receptors, also termed activin-receptor like kinases (ALK1-7) are identified in mammals. All ALKs have a similar sequence and domain structure composed of a ligand-binding extracellular domain, a transmembrane domain, and a cytoplasmic kinase domain (ten Dijke et al., 1993). The type II receptor functions to activate the type I receptor by phosphorylation of its glycine-serine rich (GS) domain. Subsequently, the type I receptor binds and phosphorylates members of the SMAD family of transcription factors. Binding of FKBP12 to the GS domain stabilizes the inactive confirmation of type I receptor and thus prevents leaky activation (Bocciardi et al., 2009; Graham and Peng, 2006).

Mouse models have shown the importance of ACVR1 for cardiac, lens and skeletal development, epithelial-mesenchymal transformation, Müllerian duct regression, generation of primordial gem cells and also for normal mesoderm formation in extraembryonic tissues at the time of gastrulation (Rajagopal et al., 2008; Yu et al., 2008) (Fukuda et al., 2006; Komatsu et al., 2007) (Wang et al., 2005) (de Sousa Lopes et al., 2004; Kaartinen et al., 2004) (Dudas et al., 2004) (Gu et al., 1999) (Mishina et al., 1999) (Ameerun et al., 1996) (Verschueren et al., 1995). Homozygous mutant mice of ACVR1 show morphological defects at E7.0 and have died by E9.5.

A single mutation of 617G-A resulting in an R206 to H conversion in the GS domain of ACVR1 is the primary cause of fibrodysplasia ossificans progressiva (FOP), a rare autosomal dominant disorder of skeletal malformations and progressive extraskeletal ossification (Shore et al., 2006). Affected individuals show heterotopic ossification in childhood, which can be induced by trauma or may occur without warning. Bone formation is episodic and progressive, leading to extra-articular ankylosis of all major joints of the axial and appendicular skeleton, rendering movement impossible. Kinase inhibitors of ACVR1 offer a potential therapeutic against FOP and have shown some efficacy in mice (Yu et al. 2008).

Here we present the structure of the ACVR1 kinase domain in complex with the imidazo[1,2-b]pyridazine inhibitor K00135a refined at 2.4 Å resolution.

Materials and Methods

ACVR1A (4DYM) Materials & Methods

Entry Clone Source: Site-directed mutagenesis

Entry Clone Accession: N/A

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

Amplified construct sequence:
CCATGGGCCACCATCATCATCATCAT
TCTTCTGGTGTAGATCTGGGTACCGA
GAACCTGTACTTCCAATCCATGCAAA
GAACAGTGGCTCACCAGATTACACTG
TTGGAGTGTGTCGGGAAAGGCAGGTA
TGGTGAGGTGTGGAGGGGCAGCTGGC
AAGGGGAAAATGTTGCCGTGAAGATC
TTCTCCTCCCGTGATGAGAAGTCATG
GTTCAGGGAAACGGAATTGTACAACA
CTGTGATGCTGAGGCATGAAAATATC
TTAGGTTTCATTGCTTCAGACATGAC
ATCAAGACACTCCAGTACCCAGCTGT
GGTTAATTACACATTATCATGAAATG
GGATCGTTGTACGACTATCTTCAGCT
TACTACTCTGGATACAGTTAGCTGCC
TTCGAATAGTGCTGTCCATAGCTAGT
GGTCTTGCACATTTGCACATAGAGAT
ATTTGGGACCCAAGGGAAACCAGCCA
TTGCCCATCGAGATTTAAAGAGCAAA
AATATTCTGGTTAAGAAGAATGGACA
GTGTTGCATAGCAGATTTGGGCCTGG
CAGTCATGCATTCCCAGAGCACCAAT
CAGCTTGATGTGGGGAACAATCCCCG
TGTGGGCACCAAGCGCTACATGGCCC
CCGAAGTTCTAGATGAAACCATCCAG
GTGGATTGTTTCGATTCTTATAAAAG
GGTCGATATTTGGGCCTTTGGACTTG
TTTTGTGGGAAGTGGCCAGGCGGATG
GTGAGCAATGGTATAGTGGAGGATTA
CAAGCCACCGTTCTACGATGTGGTTC
CCAATGACCCAAGTTTTGAAGATATG
AGGAAGGTAGTCTGTGTGGATCAACA
AAGGCCAAACATACCCAACAGATGGT
TCTCAGACCCGACATTAACCTCTCTG
GCCAAGCTAATGAAAGAATGCTGGTA
TCAAAATCCATCCGCAAGACTCACAG
CACTGCGTATCAAAAAGACTTTGACC
AAAATTGATTGACAGTAAAGGTGGAT
ACGGATCCGAATTCGAGCTCCGTCGA
CAAGCTT

Engineered R206H mutation in bold

Expressed sequence (Tag sequence in lowercase):
mghhhhhhssgvdlgtenlyfq*SMQ
RTVAHQITLLECVGKGRYGEVWRGSW
QGENVAVKIFSSRDEKSWFRETELYN
TVMLRHENILGFIASDMTSRHSSTQL
WLITHYHEMGSLYDYLQLTTLDTVSC
LRIVLSIASGLAHLHIEIFGTQGKPA
IAHRDLKSKNILVKKNGQCCIADLGL
AVMHSQSTNQLDVGNNPRVGTKRYMA
PEVLDETIQVDCFDSYKRVDIWAFGL
VLWEVARRMVSNGIVEDYKPPFYDVV
PNDPSFEDMRKVVCVDQQRPNIPNRW
FSDPTLTSLAKLMKECWYQNPSARLT
ALRIKKTLTKID

^ TEV cleavage site

Engineered R206H mutation in bold

Tags and additions: Cleavable N-terminal His6 tag

Host: SF9 Spodoptera frugiperda Insect cells

Growth Medium & Induction Protocol: Sf9 cells at a density of 2x10⁶/ml were infected with recombinant ACVR1 baculovirus (virus stock P3; 1ml of virus stock/100 ml of cell culture). Cells were shaken at 110 rpm at 27°C in an Innova shaker. After 48 hours post-infection the cultures were harvested by centrifugation for 20min at 6000rpm. Cell pellets from each 1L flask were resuspended in 15 ml binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole). Calbiochem protease inhibitor SET V was added to the cell suspension at a 1:2000 dilution and transferred to 50 ml tubes, and stored at -20°C.

Extraction buffer, extraction method: The frozen cells were thawed and the volume increased to 90 ml with binding buffer. The cells were lysed using an Emulsiflex C5 homogeniser. The cell lysate was spun down by centrifugation at 21.5K rpm and 4°C for 1 h. The supernatant was recovered for purification.

Column 1: Anion-exchange for Nucleic acid removal with DEAE cellulose (DE52, Whatmann)10 g of resin was suspended in 50 ml 1 M NaCl, and then applied onto a 2.5 x 20 cm column. The resin was then equilibrated with 50 ml binding buffer prior to loading the sample.

Column 1 Buffers:
Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole, 0.1mM TCEP
Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 25 mM imidazole, 0.1mM TCEP

Column 1 Procedure: The supernatant was first applied onto the column by gravity flow, which was followed by a wash with 50 ml wash buffer. The column flow-through and wash was directly applied onto a Ni-IDA column.

Column 2: Ni-Affinity Chromatography. 6 ml of 50 % Ni-IDA slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (25ml).

Column 2 Buffers:
Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole, 0.1mM TCEP
Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 25 mM imidazole, 0.1mM TCEP
Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 to 250 mM imidazole, 0.1mM TCEP

Column 2 Procedure: The flow-through from column 1 (DE52) was applied by gravity flow onto the Ni-IDA column. The bound protein was eluted by applying a step gradient of imidazole using 10 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM, 250 mM).

Enzymatic treatment: 0.1mg of TEV protease was added to the Ni-eluted protein to remove the tag.

Column 3: Size Exclusion Chromatography S200 HiLoad 16/60 Superdex run on ÄKTA-Express

Column 3 Buffers:
Gel filtration buffer: 150 mM NaCl, 25 mM Hepes pH 7.5

Column 3 Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. The protein was concentrated to 5 ml using an Amicon Ultra-15 filter with a 10 kDa cut-off. The concentrated protein was directly applied onto the equilibrated S200 16/60 column, and run at a flow-rate of 1 ml/min. The protein was eluted at 90-110 ml. Fractions containing the protein were pooled together.

Mass spec characterization: The purified protein was homogeneous and had an experimental mass of 34487.9 Da, as expected from primary sequences. Mass was determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% acetonitrile in water with 0.1% formic acid.

Crystallization: Protein was buffered in 25 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM DTT and 10mM L-Arg, 10mM L-Glu. To this 1mM K00135 was added and the protein concentrated to 10 mg/ml (calculated using an extinction co-efficient of 58900). Crystals were grown at 20°C in 150 nl sitting drops mixing 75 nl protein solution with 75 nl of a reservoir solution containing 1.6M MgSO₄ and 0.1M MES pH 6.5. On mounting crystals were cryoprotected with mother liquor plus 20% glycerol and flash frozen in liquid nitrogen.

Data Collection:
Resolution: 2.42Å
X-ray source: Diamond Light Source, station I02, using monochromatic radiation at wavelength 0.9795Å

References

Ameerun, R.F., de Winter, J.P., van den Eijnden-van Raaij, A.J., den Hertog, J., de Laat, S.W., and Tertoolen, L.G. (1996). Activin and basic fibroblast growth factor regulate neurogenesis of murine embryonal carcinoma cells. Cell Growth Differ 7, 1679-1688.
Bocciardi, R., Bordo, D., Di Duca, M., Di Rocco, M., and Ravazzolo, R. (2009). Mutational analysis of the ACVR1 gene in Italian patients affected with fibrodysplasia ossificans progressiva: confirmations and advancements. Eur J Hum Genet 17, 311-318.
de Sousa Lopes, S.M., Roelen, B.A., Monteiro, R.M., Emmens, R., Lin, H.Y., Li, E., Lawson, K.A., and Mummery, C.L. (2004). BMP signaling mediated by ALK2 in the visceral endoderm is necessary for the generation of primordial germ cells in the mouse embryo. Genes & development 18, 1838-1849.
Dudas, M., Sridurongrit, S., Nagy, A., Okazaki, K., and Kaartinen, V. (2004). Craniofacial defects in mice lacking BMP type I receptor Alk2 in neural crest cells. Mechanisms of development 121, 173-182.
Fukuda, T., Scott, G., Komatsu, Y., Araya, R., Kawano, M., Ray, M.K., Yamada, M., and Mishina, Y. (2006). Generation of a mouse with conditionally activated signaling through the BMP receptor, ALK2. Genesis 44, 159-167.
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Mishina, Y., Crombie, R., Bradley, A., and Behringer, R.R. (1999). Multiple roles for activin-like kinase-2 signaling during mouse embryogenesis. Developmental biology 213, 314-326.
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