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

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 EphB4⅐ephrinB2 protein interfaces and their mode of interaction, which will facilitate development of small molecule compounds inhibiting the EphB4⅐ephrinB2 interaction as novel cancer therapeutics.The protein-protein interaction between the membranebound Eph receptor tyrosine kinases with the membrane-bound ephrin ligands have now been reported in the overexpression/dysregulation in numerous tumor cell lines (1). First reported for their role in axonal guidance, this group of proteins now has defined roles in regulating several cellular processes including developmental patterning, cell attachment, and motility (2-4). The importance of these proteins in development is underscored by the fact that deletion of either the EphB4 receptor or the ephrinB2 ligand results in lethality by embryonic day 11 as a result of arrested angiogenesis but not vasculogenesis (5). Understanding the EphB4⅐ephrinB2 interaction and exploring the determinants for the unique specificity of this receptor-ligand complex is at the core of modulating this activity and will allow for a deeper understanding into the basic biology behind this interaction and for the development of novel anti-angiogenesis and anti-tumorigenesis therapeutic approaches.The EphB4 receptor and the ephrinB2 ligand are capable of transducing a signal bi-directionally into either the EphB4-expressing cell (forward signaling) or the ephrinB2-expressing cell (reverse signaling) (12, 13). Therefore, a cellular response is conducted only upon cell-cell contact. The Eph receptors are divided into two subclasses, A and B, b...

Cell–cell signaling via Eph receptors and ephrins

“…In the past year the first structure of unclompexed A-class ligand (ephrin-A5) was reported [22], as well as the structures of ephrin-B1 [23] and, most importantly, that of a third ligand/ receptor complex -EphB4/ephrin-B2 [24]. These studies further illuminate the molecular determinants of the unique binding-partner specificities of Ephs and ephrins.…”

“…Three interaction interfaces are indicated: dimerization and tetramerization -identified by crystallography [4] and confirmed by mutagenesis [25,26], as well as a potential oligomerization interface -identified by a random mutagenesis approach [26]. Schematic representation and a comparison of the interaction surfaces in the canonical intrasubclass EphB2/ephrin-B2 [20] and EphB4/ephrin-B2 [24] complexes with the unusual inter-subclass EphB2/ephrin-A5 complex [21*]. While ephrin-B2 has several interacting surface areas with the B-class receptors, the interaction of ephrin-A5 with EphB2 is mostly confined to the hydrophobic receptor surface channel.…”

“…Regarding the development of screens for small-molecule Eph/ephrin inhibitors, it should be noted that the canonical (intra-class) Eph/ephrin dimerization interfaces (EphB2/ephrin-B2 [20] and EphB4/ephrinB2 [24]) include two distinct regions, only one of which is observed in the inter-class EphB2-ephrin-A5 complex [21*]. Specifically, in addition to the Ephchannel/ephrin-loop interactions (purple on Fig.…”

Genomics of Squamous Cell Lung Cancer

“…Given that the Ephephrin family of molecules is overexpressed across a variety of cancers, several molecules that target this signaling system are currently being investigated [56 -58]. Kinase inhibitors capable of interfering with the tumor promoting effects of Ephephrin signaling, peptides and antibodies capable of inhibiting Eph-ephrin interactions, and vaccines and immunotherapies capable of facilitating tumor lysis or rejection are some of the strategies that have been described to date [67][68][69][70][71][72]. Data on the safety and efficacy of these agents in lung cancer is very limited.…”

Crystal Structure and NMR Binding Reveal That Two Small Molecule Antagonists Target the High Affinity Ephrin-binding Channel of the EphA4 Receptor

“…EphA subclass receptors remarkably differ from EphB receptors because they lack a 4-residue insert in the H-I loop of the ligand-binding domain. Previously, the structures of the EphB2 and EphB4 ligand-binding domains have been determined in both the free state and in complex with ephrins or peptide antagonists (10,11,(12)(13)(14)(15). These studies have shown that the ligand-binding domains of EphB2 and EphB4 adopt the same jellyroll ␤-sandwich architecture composed of 11 antiparallel ␤-strands connected by loops of various lengths.…”