Human MER kinase domain in complex with ADP

Target ID:

axl2

Aliases:

c-merMERMGC133349RP38

Deposition Date:

Friday 21st December 2007

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

protein-ligand

Download Coordinates:

3BRB

Authors:

Walker J.R., Huang X., Finerty P.J., Weigelt J., Arrowsmith C.H., Edwards A.M., Bochkarev A., Dhe-Paganon S.

Site:

Toronto

3BRB

tabs

Structure Details

Mer is one of three human receptor tyrosine kinases with an extracellular domain architecture composed of dual immunoglobulin and fibronectin like domains recognizing a subset of hormones including Growth Arrest Specific 6 (GAS6) (Hafizi and Dahlback, 2006). It is an essential cog in the phagocytic process, and therefore important not only during development but also critical for macrophages, retinal pigment epithelial cells, and tumorigenic tissues. Mutations in Mer have been correlated with retinitis pigmentosa (D'Cruz et al., 2000; Gal et al., 2000), and genetic knockouts lead to impaired clearance of apoptotic thymocytes (Scott et al., 2001). Elevated expression or activity occurs in ACTH-secreting adenomas (Evans et al., 2001), mantle cell lymphomas (Ek et al., 2002), and T-cell acute lymphoblastic leukemia (Graham et al., 2006; Keating et al., 2006). The correct balance of Mer is thus important to health. Here we have solved the crystal structure of human MER kinase domain in complex with ADP, which shows the details of how substrate interacts with this protein. A better understanding of the molecular functions of this class of RTK's could lead to effective disease therapies.

Materials and Methods

MERTK

PDB:3BRB

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:axl2.LIFESEQ1708542
Entry Clone Source:Open Biosystems
SGC Clone Accession:SDC037B11
Tag:N-terminal: MGSSHHHHHHSSGLVPRGS
Host:BL21 (DE3)

Construct

Prelude:
Sequence:MGSSHHHHHHSSGLVPRGSEELQNKLEDVVIDRNLLILGKILGEGEFGSVMEGNLKQEDGTSLKVAVKTMKLDNSSQREIEEFLSEAACMKDFSHPNVIRLLGVCIEMSSQGIPKPMVILPFMKYGDLHTYLLYSRLETGPKHIPLQTLLKFMVDIALGMEYLSNRNFLHRDLAARNCMLRDDMTVCVADFGLSKKIYSGDYYRQGRIAKMPVKWIAIESLADRVYTSKSDVWAFGVTMWEIATRGMTPYPGVQNHEMYDYLLHGHRLKQPEDCLDELYEIMYSCWRTDPLDRPTFSVLRLQLEKLLESLPDV
Vector:pET28a-LIC

Growth

Medium:TB with 50 µg/mL kanamycin
Antibiotics:
Procedure:A small overnight culture containing 50 µg/mL kanamycin was used to inoculate TB media containing the same concentration of antibiotics. Cultures were grown at 37°C for about 6 hours until the OD₆₀₀ reached ~0.8, the temperature was adjusted to 15°C, and expression was induced using 0.1mM IPTG overnight. Cells were harvested by centrifugation and frozen.

Purification

Procedure
Clarified supernatant was mixed with 5.0 ml 50% Talon resin slurry (Clonetech), rotated for 1 hour at 4 oC, and then loaded into a column. Ten column volumes of washing buffer were used for washing before elution with 7 mL elution buffer.

Gel-filtration was conducted using AKTAxpress (18-6645-05, GE Healthcare) with XK 16x65 columns (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare). Pre-equilibration was done with gel filtration buffer at a flow rate of 3 mL/min. 7 mL of protein sample was loaded onto the column at 1.5 mL/min, and 2 mL fractions are collected in 96-well plates (VWR 40002-012) using peak fractionation protocols with the following parameters: (Slope; min. peak width 0.833 min; level 0.000 mAU; peak start slope 10.000 AU/min; peak end slope 20.000 AU/min). Fractions were analyzed for purity using SDS-PAGE and those containing pure Mer/axl2 were pooled.

Purified protein was concentrated using 15 mL concentrators with a 10,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration of 7-10 mg/mL.

Extraction

Procedure
Frozen cell pellets obtained from 2L culture were thawed, resuspended in 50 mL lysis buffer, homogenized with Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds, and twice passed through a microfluidizer (Microfluidics M110EH) at 18,000 psi. The lysate was clarified by centrifugation (JA25.50 rotor, Avanti J-20 XPI, Beckman Coulter) for 20 minutes at 69,673 x g.
Concentration:7-10 mg/mL
Ligand
MassSpec:Calculated MW: 35841.4 g/mol
Crystallization:Mer protein (38 mg/ml) was pre-incubated with 2.5 mM ATP and 10 mM MgCl₂ for 3h at room temperature.

Crystals were obtained at 14 oC using the hanging-drop vapour-diffusion method by mixing 2 ul Mer solution with 2 ul reservoir (100 mM Tris-HCl pH 8.5, 200 mM MgCl₂, 29% PEG 400). Cryo buffer was composed of glycerol, ethylene glycol, glucose, and fructose.
NMR Spectroscopy:
Data Collection:
Data Processing:

References

D'Cruz,P.M., Yasumura,D., Weir,J., Matthes,M.T., Abderrahim,H., LaVail,M.M., and Vollrath,D. (2000). Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS rat. Hum. Mol. Genet. 9, 645-651.
Ek,S., Hogerkorp,C.M., Dictor,M., Ehinger,M., and Borrebaeck,C.A. (2002). Mantle cell lymphomas express a distinct genetic signature affecting lymphocyte trafficking and growth regulation as compared with subpopulations of normal human B cells. Cancer Res. 62, 4398-4405.
Evans,C.O., Young,A.N., Brown,M.R., Brat,D.J., Parks,J.S., Neish,A.S., and Oyesiku,N.M. (2001). Novel patterns of gene expression in pituitary adenomas identified by complementary deoxyribonucleic acid microarrays and quantitative reverse transcription-polymerase chain reaction. J. Clin. Endocrinol. Metab 86, 3097-3107.
Gal,A., Li,Y., Thompson,D.A., Weir,J., Orth,U., Jacobson,S.G., Apfelstedt-Sylla,E., and Vollrath,D. (2000). Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat. Genet. 26, 270-271.
Graham,D.K., Salzberg,D.B., Kurtzberg,J., Sather,S., Matsushima,G.K., Keating,A.K., Liang,X., Lovell,M.A., Williams,S.A., Dawson,T.L., Schell,M.J., Anwar,A.A., Snodgrass,H.R., and Earp,H.S. (2006). Ectopic expression of the proto-oncogene Mer in pediatric T-cell acute lymphoblastic leukemia. Clin. Cancer Res. 12, 2662-2669.
Hafizi,S. and Dahlback,B. (2006). Gas6 and protein S. Vitamin K-dependent ligands for the Axl receptor tyrosine kinase subfamily. FEBS J. 273, 5231-5244.
Keating,A.K., Salzberg,D.B., Sather,S., Liang,X., Nickoloff,S., Anwar,A., Deryckere,D., Hill,K., Joung,D., Sawczyn,K.K., Park,J., Curran-Everett,D., McGavran,L., Meltesen,L., Gore,L., Johnson,G.L., and Graham,D.K. (2006). Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase. Oncogene 25, 6092-6100.
Scott,R.S., McMahon,E.J., Pop,S.M., Reap,E.A., Caricchio,R., Cohen,P.L., Earp,H.S., and Matsushima,G.K. (2001). Phagocytosis and clearance of apoptotic cells is mediated by MER. Nature 411, 207-211.