Human ephrin receptor EphB4 precursor; hepatoma transmembrane kinase Homo sapiens

Target ID:

EPHB4

Aliases:

HTKMYK1TYRO11

Deposition Date:

Wednesday 11th July 2007

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

apo

Download Coordinates:

2QKQ

Authors:

Walker JR, Cuerrier D, Butler-Cole C, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S.

Site:

Toronto

ICM Datapack:

ICM Datapack

2QKQ

tabs

Structure Details

The Eph family of tyrosine kinase receptors are involved in cellular and development processes including cell migration, attraction and repulsion, and are important for developmental processes such as angiogenesis and organogenesis [1]. Their natural ligands, ephrins, result in receptor activation by inducing dimerization or clustering of receptor and consequent autophosphorylation [2].

All 14 identified members of the human Eph receptor family contain a C-terminal â€œsterile alpha motifâ€ (SAM) domain, immediately adjacent to the tyrosine kinase domain. SAM domains are conserved in over a hundred diverse protein, where they mediate homo- and heterotypic protein-protein. In a minority case, some SAM domains have evolved the ability to bind RNA interactions [3]. The SAM domain of Eph receptors, as well as in other proteins, has been shown to be capable of self-association by formation of homo- and hetero-oligomers [4, 5], and have been proposed to participate in Eph-receptor activation by mediating dimerization, or clustering of the receptor on the cytosolic side of the membrane. Moreover, the C-terminal tail of some Eph-SAM domains contain a PDZ-binding motif and can therefore further interact with other signaling protein. Interestingly, the presence of a cytoplasmic module other than the catalytic kinase domain is unique to the EPH family of receptor tyrosine kinase.

The EphB4 isoform is especially important biomedically as it is overexpressed in multiple types of tumors, where its expression levels are often correlated with increased malignancy, possibly by promoting angiogenesis. It has an intriguing role in breast cancer, where it has both tumor-suppressing and tumor-promoting activities [6].

We here present the SAM domain of the EphB4 receptor. This particular structure is a 5-helix bundle, and appears as a monomer with no extensive intermolecular contacts. The conserved Tyr924, purported to be a target of phosphorylation and mediate protein-protein interactions [7], forms a strong hydrogen bond (2.58 Ã…) with the equally conserved His957 and is partially buried, and therefore not directly susceptible to phosphorylation. This structure will help elucidate the role of SAM domains in Eph receptor regulation and the structural requirements for their oligomerization.

Materials and Methods

EPHB4

PDB:2QKQ

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_004435
Entry Clone Source:ephb4.BC052804.OBS.6584384.pOTB7
SGC Clone Accession:ephb4.896.977; plate SDC117F01
Tag:
Host:BL21 (DE3)

Construct

Prelude:
Sequence:mhhhhhhssgrenlyfq*GHPLLDQRQPHYSAFGSVGEWLRAIKMGRYEESFAAAGFGSFELVSQISAEDLLRIGVTLAGHQKKILASVQHMKSQAKPGTP
Vector:pET28-MHL

Growth

Medium:TB
Antibiotics:
Procedure:A 250 ml flask containing LB (Sigma L7658) supplemented with 50 ug/ ml kanamycin (BioShop Canada KAN 201) was inoculated from a glycerol stock of the bacteria. The flask was shaken overnight (16 hours) at 250 rpm at 37 degC.

Using the Lex system, a 2L bottle (VWR 89000-242) containing 1800 ml of TB (Sigma T0918) supplemented with 1.5% glycerol, 50 ug/ ml kanamycin and 600 ul antifoam 204 (Sigma A-8311) was inoculated with 50 ml overnight LB culture, and incubated at 37 degC. The temperature of the media was reduced to 15 degC one hour prior to induction and induced at OD600 = 6 with 100 uM isopropyl-thio-β-D-galactopyranoside (BioShop Canada IPT 001). Cultures were aerated overnight (16 hours) at 15 degC, and cell pellets collected by centrifugation and frozen at -80 degC.

Purification

Procedure
IMAC: Unclarified lysate was mixed with 2-3 mL of HisLink Protein Purification Resin (Promega V8821) per 40 mL lysate. The mixture was incubated with mixing for at least 20 minutes at 4 degC. The lysate was spun at 500 xg for 5 minutes, and the supernatant was decanted. The remaining resin was washed with 45 mL of cold wash buffer, allowing 5 minutes to settle between washes, until the supernatant was clear (usually 3-5 washes). The washed resin was transferred to a gravity column and further washed with 1 column volumes (approx. 5 mL) of wash buffer at approximately 3 mL/min. Samples were eluted from the resin by exposure to 2-3 column volumes (approx. 10 mL) of elution buffer.

His-tag cleavage: An aliquot of homemade TEV was added to the IMAC eluate to approx. 10 ug TEV / mg protein and incubated overnight at 4 degC prior to gel filtration.

Gel filtration chromatography: An XK 16x65 column (part numbers 18-1031-47 and 18-6488-01, GE Healthcare) packed with HighLoad Superdex 200 resin (10-1043-04, GE Healthcare) was pre-equilibrated with gel filtration buffer for 1.5 column volumes using an AKTAxpress (18-6645-05, GE Healthcare) at a flow rate of 3 mL/min. The eluate sample from the IMAC step (approx. 10 mL) was loaded onto the column at 1.5 mL/min, and 2mL fractions were collected into 96-well plates (VWR 40002-012) using peak fractionation protocols). Fractions observed by a UV absorption chromatogram to contain the protein were pooled.

Removal of TEV / uncleaved protein: A 0.5 mL aliquot of His-link resin was added to the pooled fractions along with imidazole to a final concentration of 20 mM. This sample was mixed at 4 degC for 20 min, and the resin collected in a gravity column prior to proceeding with concentration.

Extraction

Procedure
Frozen cell pellet contained in bags (Beckman 369256) obtained from 2L of culture were thawed by soaking in warm water. Each cell pellet was resuspended in 25-40 mL lysis buffer and homogenized using an Ultra-Turrax T8 homogenizer (IKA Works) at maximal setting for 30-60 seconds per pellet. Cell lysis was accomplished by sonication (Virtis408912, Virsonic) on ice: the sonication protocol was 10 sec pulse at half-maximal frequency (5.0), 10 second rest, for 10 minutes total sonication time per pellet.
Concentration:The purified protein was concentrated using a 15 mL concentrator with a 5,000 molecular weight cut-off (Amicon Ultra-15, UFC900524, Millipore) at 3750 rpm, typically resulting in a final concentration of 10-15 mg/mL. The imidazole from the previous step was diluted by performing at least one cycle of buffer exchange in the concentrator using gel filtration buffer.
Ligand
MassSpec:
Crystallization:Crystals of the EphB4 SAM domain were grown at 298 K using the sitting drop method by mixing equal volumes of 2.3 M NaKPO₄, pH 6.4 and 13 mg/mL protein. The crystals were cryoprotected by transferring the crystals to a drop containing mother liquor to which glycerol was added to 20%.
NMR Spectroscopy:
Data Collection:
Data Processing:

References