Structural and Biophysical Characterization of the EphB4·EphrinB2 Protein-Protein Interaction and Receptor Specificity

Michael I. Recht,Jan Marcus Seifert,Gye Won Han,Michelle L. Kraus,Jill E. Chrencik,Peter Kuhn,Anand R. Kolatkar,Hans Widmer,Manfred Auer,Alexei Brooun

doi:10.1074/jbc.m605766200

Abstract

Increasing evidence implicates the interaction of the EphB4 receptor with its preferred ligand, ephrinB2, in pathological forms of angiogenesis and in tumorigenesis. To identify the molecular determinants of the unique specificity of EphB4 for ephrinB2, we determined the crystal structure of the ligand binding domain of EphB4 in complex with the extracellular domain of ephrinB2. This structural analysis suggested that one amino acid, Leu-95, plays a particularly important role in defining the structural features that confer the ligand selectivity of EphB4. Indeed, all other Eph receptors, which promiscuously bind many ephrins, have a conserved arginine at the position corresponding to Leu-95 of EphB4. We have also found that amino acid changes in the EphB4 ligand binding cavity, designed based on comparison with the crystal structure of the more promiscuous EphB2 receptor, yield EphB4 variants with altered binding affinity for ephrinB2 and an antagonistic peptide. Isothermal titration calorimetry experiments with an EphB4 Leu-95 to arginine mutant confirmed the importance of this amino acid in conferring high affinity binding to both ephrinB2 and the antagonistic peptide ligand. Isothermal titration calorimetry measurements also revealed an interesting thermodynamic discrepancy between ephrinB2 binding, which is an entropically driven process, and peptide binding, which is an enthalpically driven process. These results provide critical information on the EphB4*ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4*ephrinB2 interaction as novel cancer therapeutics.

Highlights

Ephrin ligands have been reported in the overexpression/dysregulation in numerous tumor cell lines [1]
Given that the EphB41⁄7ephrinB2 interaction is important in angiogenesis and that EphB4 is overexpressed in several tumor types (1, 20 –22), modulating this protein-protein interaction is a potential approach to slowing tumor angiogenesis and tumor growth
A multiple sequence alignment with members of the EphB subclass reveals that the EphB4 receptor lacks a conserved arginine and instead contains a leucine

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The human EphB4 receptor [17–196] was expressed and purified in insect cells as described elsewhere [19]. Crystallization, Data Collection, and Structure Solution—The EphB41⁄7ephrinB2 complex was concentrated to 20 mg/ml in a buffer containing 25 mM Tris, pH 7.8, 150 mM NaCl, and 5 mM CaCl2 and crystallized by sitting drop vapor diffusion at 20 °C against a precipitant of 2.2 M ammonium sulfate and 100 mM Tris, pH 7.8. EphB4 (wild type or mutant) was present in the sample cell at a concentration of 12–15 ␮M, and the injection syringe contained either 127 ␮M ephrinB2 or 200 ␮M TNYL-RAW. Titrations of TNYL-RAW with the L95R mutant of EphB4 were performed with 2 mM TNYL-RAW in the injection syringe and 15 ␮M EphB4 (L95R) in the sample cell Data for these titrations were fit assuming a stoichiometry of 1, and at least 60% saturation at the final peptide concentration as described [19, 34]. The calculated Z-factor for 108 samples, each at 2 different protein concentrations representing upper and lower plateaus of the dose response curve, is 0.715

RESULTS

Number of atoms Protein Solvent

DISCUSSION

Kd nM