Kinase domain of human VEGFR1

Target ID:

FLT1A

Deposition Date:

Sunday 31st May 2009

Families:

Protein Kinase

Related Diseases:

Cancer

Structure type:

protein-ligand

Download Coordinates:

3HNG

Authors:

Tresaugues, L., Roos, A., Arrowsmith, C.H., Berglund, H., Bountra, C., Collins, R., Edwards, A.M., Flodin, S., Flores, A., Graslund, S., Hammarstrom, M., Johansson, A., Johansson, I., Karlberg, T., Kotyenova, T., Kotzch, A., Moche, M., Nielsen, T.K., Nyman, T., Persson, C., Sagemark, J., Schueler, H., Schutz, P., Siponen, M.I., Svensson, L., Thorsell, A.G., Van Den Berg, S., Weigelt, J., Welin, M., Wisniewska, M., Nordlund, P. Structural Genomics Consortium (SGC)

Site:

Stockholm

3HNG

tabs

Structure Details

Vascular endothelial growth factor receptor 1 (VEGFR-1, FLT-1) is a receptor tyrosine kinase (RTK) which belongs to a family of type III receptor tyrosine kinase. This family includes the colony-stimulating factor 1 receptor, FLT-3 receptor, FLT-4 (VEGFR-3), VEGFR-2, the platelet-derived growth factor α and β and c-Kit. The characteristics of this family are: i) an extracellular domain composed by multiple copies of immunoglobulin domains (7 for both FLT-1 and VEGFR-2), ii) a single transmembrane helix, iii) an autoinhibitory juxtamembrane domain and iv) a kinase insertion domain (KID) which disrupts the C-lobe of the catalytic kinase domain [1, 2].
VEGFR-1 can both promote or inhibit angiogenesis [3, 4] and is thus involved in angiogenesis related pathology as cancer or retinopathy of prematurity [5, 6].
Signalling through VEGFR-1 is linked to binding to three different ligands : PLGF, VEGF-A and VEGF-B. Actually the proposed model of VEGFR-1-mediated regulation could be initiated in three different way of action : i) VEGF-trapping by capturing VEGFR-2 ligand thus antagonizing VEGFR-2 signaling [7], ii) heterodimerization with VEGFR-2 [8] and iii) homodimerization [8].

We have solved and refined to 2.7Å the structure of the catalytical domain of unphosphorylated VEGFR-1 complexed with N-(4-Chlorophenyl)-2-[(pyridin-4-ylmethyl)amino]benzamide, an inhibitor with a scaffold based upon anthranilic acid [9] (PDB entry: 3HNG). The protein construct used for crystallization encompasses residues 801 to 1158 from human VEGFR-1 including 27 residues from the juxtamembrane domain and the full-length KID. Dataset was collected on ESRF beamline ID14-2 and structure was solved by molecular replacement using the structure of the catalytical domain of VEGFR2 in complex with a benzimidazole inhibitor (PDB entry : 2QU5) [10]. The model has been refined to lead to R and Rfree values of respectively 19.4% and 26%.

VEGFR-1 adopts the classic kinase fold with a N-lobe constituted mostly by β-sheets and a helical C-lobe. The structure shows VEGFR-1 in the inhibited state previously described for this family of protein [11] in where the activation loop is in a "closed conformation" between the N- and C- lobe. Thus, Tyr1053 is located in the active site playing the role of a pseudo- substrate. This unactive state is maintained by the inhibitor which restrained the DFG motif in a conformation very similar to one produced by juxtamembrane autoinhibiton.
If 26 residues of the juxtamembrane domain are clearly visible in the density, position of only 7 out of 72 residues of the KID could be attributed due to the intrinsic disorder of this region.

Materials and Methods

FLT1

PDB:3HNG

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:gi|24660372
Entry Clone Source:SGC Toronto. Synthetic gene
SGC Clone Accession:FLT1A-s002
Tag:N-terminal hexahistidine tag with integrated TEV protease cleavage site: mhhhhhhssgvdlgtenlyfq*sm
Host:E.coli BL21(DE3) R3 pRARE, where R3 denotes a derivative of BL21(DE3) resistant to a strain of T1 bacteriophage (SGC Oxford) and the pRARE plasmid originating from the Rosetta strain (Novagen) supplies tRNAs for rare codons.

Construct

Prelude:
Sequence:mhhhhhhssgvdlgtenlyfqsmPDEVPLDEQCERLPYDASKWEFARERLKLGKSLGRGAFGKVVQASAFGIKKSPTCRTVAVKMLKEGATASEYKALMTELKILTHIGHHLNVVNLLGACTKQGGPLMVIVEYCKYGNLSNYLKSKRDLFFLNKDAALHMEPKKEKMEPGLEQGKKPRLDSVTSSESFASSGFQEDKSLSDVEEEEDSDGFYKEPITMEDLISYSFQVARGMEFLSSRKCIHRDLAARNILLSENNVVKICDFGLARDIYKNPDYVRKGDTRLPLKWMAPESIFDKIYSTKSDVWSYGVLLWEIFSLGGSPYPGVQMDEDFCSRLREGMRMRAPEYSTPEIYQIMLDCWHRDPKERPRFAELVEKLGDLL
Vector:pNIC-Bsa4

Growth

Medium:
Antibiotics:
Procedure:Cells from a glycerol stock were grown in 2 x 50 ml TB supplemented with 8 g/l glycerol, 100 µg/ml kanamycin and 34 µg/ml chloramphenicol at 37 °C overnight. The overnight culture (100 ml) was used to inoculate 9 l TB (divided into 6 x 1.5 l bottles) supplemented with 8 g/l glycerol, 50 µg/ml kanamycin and approximately 200 µl 204 Antifoam A6426 (Sigma) per bottle. The culture was grown in a LEX bioreactor system (Harbinger Biotechnology) at 37 °C until OD600nm had reached 1.6. The culture was down-tempered to 18 °C over a period of 1 hour before target expression was induced by addition of 0.5 mM IPTG. Expression was allowed to continue overnight and cells were harvested the following morning by centrifugation (4,400 x g, 10 min, 4 °C). The resulting cell pellet (152 g wet cell weight) was then stored at -80 °C.

Purification

Procedure
Columns
IMAC: Ni-charged 5 ml HisTrap HP (GE Healthcare)
Gel filtration column: HiLoad 16/60 Superdex 75 Prep Grade (GE Healthcare)
Ion exchange column : MonoQ 5/50 GL (GE Healthcare)

Procedure
The whole purification procedure was performed at 4 ºC. Protein samples were loaded onto three separates IMAC columns previously equilibrated in Lysis buffer. Each IMAC column was then washed with, first, 100 ml of IMAC wash1 buffer followed by 100 ml of IMAC wash2 buffer. These operations were carried on using a peristaltic pump. The three IMAC columns were then connected in series on an ÄKTA Prime system (GE Healtchare). Bound protein was eluted from the IMAC columns with IMAC elution buffer then loaded onto the gel filtration column equilibrated in GF buffer. Fractions containing the target protein were pooled.

Tag removal and unphosphorylation
Proteolytic removal of the N-terminal histidine tag and unphosphorylation were performed in the same step. Protein sample was incubated with His-tagged TEV protease in a molar ratio of 10:1, 500 U of Calf Intestine Alkaline Phosphatase (CIAP) (Fermentas) and 10 mM MgCl2 at 4 ºC overnight under agitation. The proteolytic reaction went to completion, as judged by SDS-PAGE. The reaction mix was supplemented with 20mM imidazole and incubated with 3 ml Ni-NTA resin (Qiagen) equilibrated in TEV purification buffer. The sample was loaded onto an empty gravity flow column (Qiagen) and the flowthrough was collected.

Ion exchange
The sample was concentrated to 5 ml in a Vivaspin 10,000 MWCO (Sartorius) then diluted to 20 ml in MonoQ low salt 7.5 buffer. This step was done twice. The protein was loaded onto the ion exchange column equilibrated in MonoQ low salt pH 8.5 buffer. A gradient between MonoQ low salt pH 8.5 buffer and MonoQ high salt pH 8.5 buffer was applied onto the column. Fractions eluted between 7 and 9% of MonoQ high salt pH 8.5 buffer were pooled, supplemented with 200 mM NaCl and concentrated to 14mg/ml in a Vivaspin 10,000 MWCO (Sartorius). Identity and level of phosphorylation of FLT1 were assessed by mass spectrometry.

Extraction

Procedure
The pellet was splitted into three equal parts. Each part was resuspended into a final volume of 150ml of Lysis buffer (including the volume of the pellet) supplemented with a knife edge of lysozyme (Fluka), one tablet of Complete EDTA-free protease inhibitor (Roche Applied Science) and 1000 U Benzonase (Merck). The samples are left under agitation for 1 h at 4 C. Cells were disrupted by sonication (Vibra-Cell, Sonics) at 80% amplitude for 6 min effective time (pulsed 4s on, 4s off) and cell debris was removed by centrifugation (49,000 x g, 40 min, 4 ºC). The supernatant was decanted and filtered through a 0.45 µm flask filter.
Concentration:
Ligand
MassSpec:
Crystallization:Protein sample was incubated for 30 min at room temperature with 2mM N-(4-Chlorophenyl)-2-((pyridin-4-ylmethyl)amino)benzamide (Calbiochem) dissolved in DMSO. The sample was then spinned for 30 min at 20,000 x g in a table-top centrifuge. Supernatant was used to set-up crystallization plates. Crystals were obtained by the sitting drop vapour diffusion method in a 96-well plate. 0.1 µl protein solution (11.2 mg/ml) was mixed with 0.2 µl of well solution consisting of 0.2 M potassium formate and 20% PEG 3350. The plate was incubated at 4 ºC and 230 µm long thin plates appeared between 14 and 28 days. The crystals were quickly transferred to a cryo solution consisting of 0.2 M potassium formate, 22% PEG 3350, 0.3 M NaCl, 30% ethylene-glycol and flash frozen in liquid nitrogen.
NMR Spectroscopy:
Data Collection:Diffraction data to 2.7 Å resolution was collected at ESRF beamline ID14-2.
Data Processing:The structure was solved by molecular replacement using the structure of human VEGFR2 kinase domain as template (PDB: 2QU5). The space group was I222 with cell dimensions a=58.04 Å, b=71.15 Å c=196.02 Å. The structure was solved using the Phenix suite of program. One monomer was located in the asymmetric unit. Iterative cycles of manual improvement and refinement were made respectively in Coot and Refmac. Data in the interval 44.99-2.7 Å resolution was used and at the end of the refinement the R values were: R=19.7% and Rfree=26.0%. Coordinates for the crystal structure were deposited in the Protein Data Bank, accession code 3HNG.

References

Yarden, Y., et al., Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature, 1986. 323(6085): p. 226-32.
Ullrich, A. and J. Schlessinger, Signal transduction by receptors with tyrosine kinase activity. Cell, 1990. 61(2): p. 203-12.
Fong, G.H., et al., Role of the Flt-1 receptor tyrosine kinase in regulating the assembly of vascular endothelium. Nature, 1995. 376(6535): p. 66-70.
Fong, G.H., et al., Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development, 1999. 126(13): p. 3015-25.
Shih, S.C., et al., Selective stimulation of VEGFR-1 prevents oxygen-induced retinal vascular degeneration in retinopathy of prematurity. J Clin Invest, 2003. 112(1): p. 50-7.
Wey, J.S., et al., Vascular endothelial growth factor receptor-1 promotes migration and invasion in pancreatic carcinoma cell lines. Cancer, 2005. 104(2): p. 427-38.
Hiratsuka, S., et al., Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci U S A, 1998. 95(16): p. 9349-54.
Rahimi, N., V. Dayanir, and K. Lashkari, Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells. J Biol Chem, 2000. 275(22): p. 16986-92.
Furet, P., et al., Identification of a new chemical class of potent angiogenesis inhibitors based on conformational considerations and database searching. Bioorg Med Chem Lett, 2003. 13(18): p. 2967-71.
Potashman, M.H., et al., Design, synthesis, and evaluation of orally active benzimidazoles and benzoxazoles as vascular endothelial growth factor-2 receptor tyrosine kinase inhibitors. J Med Chem, 2007. 50(18): p. 4351-73.
Griffith, J., et al., The structural basis for autoinhibition of FLT3 by the juxtamembrane domain. Mol Cell, 2004. 13(2): p. 169-78.