Human ephrin receptor EPHA2 in complex with ephrin A1 isoform a precursor

Target ID:

EPHA2

Aliases:

ECK

Deposition Date:

Wednesday 30th April 2008

Families:

Protein Kinase

Related Diseases:

CancerSignalling

Structure type:

protein-protein

Download Coordinates:

3CZU

Authors:

Walker JR, Yermekbayeva L, Seitova A, Butler-Cole C, Bountra C, Wikstrom M, Arrowsmith CH, Edwards AM, Bochkarev A, Dhe-Paganon S

Site:

Toronto

ICM Datapack:

ICM Datapack

3CZU

tabs

Structure Details

This gene belongs to the ephrin receptor subfamily of the protein-tyrosine kinase family. EPH and EPH-related receptors have been implicated in mediating developmental events, particularly in the nervous system. Receptors in the EPH subfamily typically have a single kinase domain and an extracellular region containing a Cys-rich domain and 2 fibronectin type III repeats. The ephrin receptors are divided into 2 groups based on the similarity of their extracellular domain sequences and their affinities for binding ephrin-A and ephrin-B ligands. This gene encodes a protein that binds ephrin-A ligands. [provided by RefSeq]

Here we report the crystal structure of the complex formed between the ligand binding domain of EphA2 and the ligand ephrin-A1. The EphA2 receptor binds an ephrin ligand by the canonical insertion of an extended ephrin loop into a channel at the surface of the receptor. An induced fit model is proposed, supported by comparison of the complex and Apo structures of the LBD (3C8X). The EphA2 147-163 loop becomes ordered upon binding ligand, and the loop 55-66 also shifts position upon binding to ephrin-A1. Finally, higher-order interactions seen in the crystal may be physiologically relevant, potentially revealing a new mechanism to promote clustering and the initiation of bidirectional signaling.

Materials and Methods

EPHA2-EFNA1 complex

PDB:3CZU

Revision

Revision Type:created
Revised by:created
Revision Date:created
Entry Clone Accession:NP_004422.2
Entry Clone Source:epha2.BC037166.MGC.AU80A3.pOTB7
efna1a.BC032698.MGC.AT54G10.pCMVSPORT6
SGC Clone Accession:epha2.023.202.121A05 (SDC121A05)
efna1a.017.171. 121F08 (SDC121A05)
Tag:N-terminal: APEHHHHHHDYDIPTTENLYFQGAMD for both EphA2 and efna1a.
Host:Sf9 insect cells

Construct

Prelude:
Sequence:EphA2:
aapehhhhhhdydipttenlyfqgamdAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCP

efna1a:
aapehhhhhhdydipttenlyfqgamdAADRHTVFWNSSNPKFRNEDYTIHVQLNDYVDIICPHYEDHSVADAAMEQYILYLVEHEEYQLCQPQSKDQVRWQCNRPSAKHGPEKLSEKFQRFTPFTLGKEFKEGHSYYYISKPIHQHEDRCLRLKVTVSGKITHSPQAHVNPQEKRLAADDP
Vector:pFHMSP-LIC-N for both EphA2 and efna1a.

Growth

Medium:
Antibiotics:
Procedure:Plasmid transfer vector pFHMSP-LIC-N containing the gene was transformed into DH10Bac E.coli cells (Invitrogen) to obtain recombinant viral DNA. SF9 cells were transfected with Bacmid DNA using Cellfectin reagent (Invitrogen), and recombinant baculovirus was generated. Viral stock was amplified from P1 to P3.

Protein production: Sf9 cells grown in HyQ® SFX Insect Serum Free Medium (Cat.# SH3027802) at density of 3.5 million cells per milliliter of media and with viability not less then 97 % were infected with 5 mL of P3 viral stock for each 1 L of cell culture. Cell culture medium was collected after 4 days of incubation on a shaker at 100 RPM and 27 °C when cells viability dropped to 25-45 %.

Purification

Procedure
IMAC purification: A 3.2 L volume of medium was mixed with 30 ml pre-equilibrated NiNTA Superflow beads and stirred (Talboys/Troemner) for 1 hour. The resin was transferred to a 100 ml gravity column, washed with 300 ml of Washing Buffer, and the protein was eluted with 90 ml of Elution Buffer. A second round of NiNTA batch absorption may have been performed for increased yield. The eluate was dialyzed against 50-100 X volume of Buffer A overnight at 4 degC.

Extraction

Procedure
The cultured medium was centrifuged at 14,000 xg for 15 minutes, and the pH of the supernatant was adjusted to 7.5 at room temperature by adding 10x Buffer_A. Protease inhibitors were added to final concentrations of 1 mM (phenylmethanesulfonyl fluoride, PMSF, Bioshop) and 2 mM (benzamidine hydrochloride, Sigma).
Concentration:Purified protein was concentrated using 15 mL concentrators with an appropriate molecular weight cut-off (Amicon Ultra-15 10,000 MWCO, Millipore) to a final value of 10 mg/mL. Average yield was about 2 mg/L.
Ligand
MassSpec:
Crystallization:Mixture of EphA2 (at 9.6 g/L) and efna1a (at 22 g/L) in ratio 0.02 uM : 0.02 uM were set (500 nL protein + 500 nL well solution) at 18 ºC. Optimal crystallization conditions centered at 14.9% PEG 4000, 0.1 M Sodium Citrate pH 5.6, 20% Isopropanol. Mix4 was used as cryoprotectant for data-collection at 100 ºK.
NMR Spectroscopy:
Data Collection:
Data Processing: