Abstract
The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of approximately 15 microm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-A resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities.
Highlights
The EphB2 receptor has been recently found to be overexpressed in many types of cancer, including gastric [12, 13], colorectal (14 –18), ovarian [19], breast [20], and prostate cancers [21, 22], and glioblastoma [23, 24]
The binding interfaces for each peptide with the receptor are uniquely distinct, despite the fact the ligand-binding domains of the EphB2 and EphB4 receptors share 45% sequence identity. Both peptides sterically inhibit the interaction of the erythropoietin-producing hepatocellular carcinoma (Eph) receptor with the ephrin G–H loop, and antagonize ephrin binding
It appears that a general requirement for effective peptide binding is the formation of an interaction network capable of mediating the conformational stability of the flexible loops of the Eph receptor, and the J–K loop
Summary
The ligand-binding domain of the Eph receptor has been characterized as the minimal region required for high affinity interaction with the ephrins [29]. Reported crystal structures of Eph receptors in complex with a ligand revealed a heterodimerization interface on the surface of the Eph receptor ligand-binding domain that mediates a high affinity receptor/ligand interaction. The crystal structure of the EphB4 receptor in complex with the high affinity (40 nM) and antagonistic TNYL-RAW (TNYLFSPNGPIARAW) peptide was elucidated [31]. Phage display screens have identified a series of 12-mer peptides that target the ligand-binding domains of several Eph receptors and antagonize their interactions with ephrins, whereas maintaining exceptional specificity for a particular Eph receptor [32]. Thermodynamic characterization of the EphB2 ligand-binding domain-SNEW complex revealed a low micromolar binding affinity, consistent with the weak interaction network observed in the crystal structure. Together with previously available structural information, our data should accelerate drug discovery efforts aimed at targeting the extracellular Eph/ephrin protein-protein interaction
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