Human ACVR1 kinase in complex with LDN-213844

Target ID:

ACVR1A

Aliases:

ACTRIACVR1ACVR1AACVRLK2ALK2FOPSKR1TSRI

Deposition Date:

Tuesday 26th March 2013

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

protein-ligand

Download Coordinates:

4BGG

Authors:

C. Sanvitale, P. Canning, C.D.O. Cooper, Y. Wang, A.H. Mohedas, S. Choi, P.B. Yu, G.D. Cuny, R. Nowak, D. Coutandin, M. Vollmar, F. von Delft, C.H. Arrowsmith, A.M. Edwards, C. Bountra, A. Bullock, Structural Genomics Consortium (SGC)

Site:

Oxford

4BGG

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 inhibitor LDN-213844 refined at 2.56 Å resolution.

LDN-213844

Materials and Methods

ACVR1

PDB:4BGG

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:
Entry Clone Source:Site-directed mutagenesis
SGC Clone Accession:ACVR1A-c096
Tag:MGHHHHHHSSGVDLGTENLYFQ*SM. cleavable N-terminal hexahistidine tag.
Host:SF9 Spodoptera frugiperda Insect cells

Construct

Prelude:
Sequence:MGHHHHHHSSGVDLGTENLYFQSMQRTVARDITLLECVGKGRYGEVWRGSWQGENVAVKIFSSRDEKSWFRETELYNTVMLRHENILGFIASDMTSRHSSTQLWLITHYHEMGSLYDYLQLTTLDTVSCLRIVLSIASGLAHLHIEIFGTQGKPAIAHRDLKSKNILVKKNGQCCIADLGLAVMHSQSTNQLDVGNNPRVGTKRYMAPEVLDETIQVDCFDSYKRVDIWAFGLVLWEVARRMVSNGIVEDYKPPFYDVVPNDPSFEDMRKVVCVDQQRPNIPNRWFSDPTLTSLAKLMKECWYQNPSARLTALRIKKTLTKIDEngineered Q207D mutation in bold .
Vector:pFB-LIC-Bse

Growth

Medium:Sf9 cells at a density of 2x106/ml were infected with recombinant ACVR1 baculovirus (virus stock P3; 1ml of virus stock/1000 ml of cell culture). Cells were shaken at 110 rpm at 27Â°C in an Innova shaker. After 72 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 -20Â°C.
Antibiotics:
Procedure:

Purification

Buffers
Procedure

Extraction

Buffers
Procedure
Extraction buffer, extraction method: The frozen cells were thawed and the volume increased to 80 ml with binding buffer. The cells were lysed by sonication over 12 min with the sonicator pulsing ON for 5 sec and OFF for 10 sec. The DNA was precipitated using 0.15% PEI (polyethyleneimine) pH 8. The cell lysate was spun down by centrifugation at 21.5K rpm at 4°C for 1 h. The supernatant was recovered for purification.Column 1: Ni-Affinity Chromatography. 6 ml of 50 % Ni-sepharose slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (25ml). 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 TCEPElution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 to 250 mM imidazole, 0.1mM TCEPProcedure: The supernatant was applied by gravity flow onto the Ni-sepharose column. The bound protein was eluted by applying a step gradient of imidazole Â 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 2: Size Exclusion Chromatography Â S200 HiLoad 16/60 Superdex run on ÄKTA-ExpressBuffer: Gel Filtration buffer: 300 mM NaCl, 50 mM Hepes pH 7.5, 0.5mM TCEPProcedure: 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 90-108 ml. Fractions containing the protein were pooled together. Column 3: Ni-Affinity Chromatography. 0.75 ml of 50 % Ni-sepharose slurry was applied onto a 1.5 x 10 cm column. The column was equilibrated with binding buffer (15ml).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 TCEPElution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 250 mM imidazole, 0.1mM TCEPProcedure: The cleaved protein was passed through the column followed by 3ml binding buffer. It was then washed with 8ml wash buffer. Anything remaining bound to the column was eluted with 15ml elution buffer.
Concentration:
Ligand
MassSpec:The purified protein was homogeneous and had an experimental mass of 34492.3 (after TEV cleavage), closely matching the expected mass 34492.7. 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% isopropanol in water with 0.1% formic acid.
Crystallization:Protein was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM DTT and 10mM L-arginine, 10 mM L-glutamate. The protein was concentrated to 10 mg/ml (calculated using an extinction co-efficient of 58900) in the presence of the inhibitor LDN-213844 (1 mM end concentration). Crystals were grown at 20°C in 150 nl sitting drops mixing 100 nl protein solution with 50 nl of a reservoir solution containing 0.2M ammonium citrate and 20% PEG 3350. On mounting crystals were cryoprotected with mother liquor plus 20% ethylene glycol before transfer to liquid nitrogen.
NMR Spectroscopy:
Data Collection:Resolution: 2.56 Ã resolutionX-ray source: Diamond Light Source, station I04-1
Data Processing:

References

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