Sizes of interface residues account for cross‐class binding affinity patterns in Eph receptor–ephrin families

Eph receptors comprise the largest known family of receptor tyrosine kinases in mammals. They bind members of a second family, the ephrins. As both Eph receptors and ephrins are membrane bound, interactions permit unusual bidirectional cell–cell signaling. Eph receptors and ephrins each form two classes, A and B, based on sequences, structures, and patterns of affinity: Class A Eph receptors bind class A ephrins, and class B Eph receptors bind class B ephrins. The only known exceptions are the receptor EphA4, which can bind ephrinB2 and ephrinB3 in addition to the ephrin‐As (Bowden et al., Structure 2009;17:1386–1397); and EphB2, which can bind ephrin‐A5 in addition to the ephrin‐Bs (Himanen et al., Nat Neurosci 2004;7:501–509). A crystal structure is available of the interacting domains of the EphA4‐ephrin B2 complex (wwPDB entry 2WO2) (Bowden et al., Structure 2009;17:1386–1397). In this complex, the ligand‐binding domain of EphA4 adopts an EphB‐like conformation. To understand why other cross‐class EphA receptor–ephrinB complexes do not form, we modeled hypothetical complexes between (1) EphA4–ephrinB1, (2) EphA4–ephrinB3, and (3) EphA2–ephrinB2. We identify particular residues in the interface region, the size variations of which cause steric clashes that prevent formation of the unobserved complexes. The sizes of the sidechains of residues at these positions correlate with the pattern of binding affinity. Proteins 2014; 82:349–353. © 2013 Wiley Periodicals, Inc.     

EphrinB3 blocks EphB3 dependence receptor functions to prevent cell death following traumatic brain injury

Eph receptor tyrosine kinases and their membrane-bound ligands, ephrins, have a variety of roles in the developing and adult central nervous system that require direct cell–cell interactions; including regulating axon path finding, cell proliferation, migration and synaptic plasticity. Recently, we identified a novel pro-survival role for ephrins in the adult subventricular zone, where ephrinB3 blocks Eph-mediated cell death during adult neurogenesis. Here, we examined whether EphB3 mediates cell death in the adult forebrain following traumatic brain injury and whether ephrinB3 infusion could limit this effect. We show that EphB3 co-labels with microtubule-associated protein 2-positive neurons in the adult cortex and is closely associated with ephrinB3 ligand, which is reduced following controlled cortical impact (CCI) injury. In the complete absence of EphB3 (EphB3^−/−), we observed reduced terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL), and functional improvements in motor deficits after CCI injury as compared with wild-type and ephrinB3^−/− mice. We also demonstrated that EphB3 exhibits dependence receptor characteristics as it is cleaved by caspases and induces cell death, which is not observed in the presence of ephrinB3. Following trauma, infusion of pre-clustered ephrinB3-Fc molecules (eB3-Fc) into the contralateral ventricle reduced cortical infarct volume and TUNEL staining in the cortex, dentate gyrus and CA3 hippocampus of wild-type and ephrinB3^−/− mice, but not EphB3^−/− mice. Similarly, application of eB3-Fc improved motor functions after CCI injury. We conclude that EphB3 mediates cell death in the adult cortex through a novel dependence receptor-mediated cell death mechanism in the injured adult cortex and is attenuated following ephrinB3 stimulation.     

Rac-GAP alpha-chimerin regulates motor-circuit formation as a key mediator of EphrinB3/EphA4 forward signaling

The ephrin/Eph system plays a central role in neuronal circuit formation; however, its downstream effectors are poorly understood. Here we show that alpha-chimerin Rac GTPase-activating protein mediates ephrinB3/EphA4 forward signaling. We discovered a spontaneous mouse mutation, miffy (mfy), which results in a rabbit-like hopping gait, impaired corticospinal axon guidance, and abnormal spinal central pattern generators. Using positional cloning, transgene rescue, and gene targeting, we demonstrated that loss of alpha-chimerin leads to mfy phenotypes similar to those of EphA4(-/-) and ephrinB3(-/-) mice. alpha-chimerin interacts with EphA4 and, in response to ephrinB3/EphA4 signaling, inactivates Rac, which is a positive regulator of process outgrowth. Moreover, downregulation of alpha-chimerin suppresses ephrinB3-induced growth cone collapse in cultured neurons. Our findings indicate that ephrinB3/EphA4 signaling prevents growth cone extension in motor circuit formation via alpha-chimerin-induced inactivation of Rac. They also highlight the role of a Rho family GTPase-activating protein as a key mediator of ephrin/Eph signaling.     

Small molecules can selectively inhibit ephrin binding to the EphA4 and EphA2 receptors

The erythropoietin-producing hepatocellular (Eph) family of receptor tyrosine kinases regulates a multitude of physiological and pathological processes. Despite the numerous possible research and therapeutic applications of agents capable of modulating Eph receptor function, no small molecule inhibitors targeting the extracellular domain of these receptors have been identified. We have performed a high throughput screen to search for small molecules that inhibit ligand binding to the extracellular domain of the EphA4 receptor. This yielded a 2,5-dimethylpyrrolyl benzoic acid derivative able to inhibit the interaction of EphA4 with a peptide ligand as well as the natural ephrin ligands. Evaluation of a series of analogs identified an isomer with similar inhibitory properties and other less potent compounds. The two isomeric compounds act as competitive inhibitors, suggesting that they target the high affinity ligand-binding pocket of EphA4 and inhibit ephrin-A5 binding to EphA4 with K(i) values of 7 and 9 mum in enzyme-linked immunosorbent assays. Interestingly, despite the ability of each ephrin ligand to promiscuously bind many Eph receptors, the two compounds selectively target EphA4 and the closely related EphA2 receptor. The compounds also inhibit ephrin-induced phosphorylation of EphA4 and EphA2 in cells, without affecting cell viability or the phosphorylation of other receptor tyrosine kinases. Furthermore, the compounds inhibit EphA4-mediated growth cone collapse in retinal explants and EphA2-dependent retraction of the cell periphery in prostate cancer cells. These data demonstrate that the Eph receptor-ephrin interface can be targeted by inhibitory small molecules and suggest that the two compounds identified will be useful to discriminate the activities of EphA4 and EphA2 from those of other co-expressed Eph receptors that are activated by the same ephrin ligands. Furthermore, the newly identified inhibitors represent possible leads for the development of therapies to treat pathologies in which EphA4 and EphA2 are involved, including nerve injuries and cancer.     

EphB receptors and ephrinB ligands: regulators of vascular assembly and homeostasis

Eph receptors comprise the largest family of receptor tyrosine kinases consisting of eight EphA receptors (with five corresponding glycosyl-phosphatidyl-inositol-anchored ephrinA ligands) and six EphB receptors (with three corresponding transmembrane ephrinB ligands). Originally identified as neuronal pathfinding molecules, genetic loss of function experiments have identified EphB receptors and ephrinB ligands as crucial regulators of vascular assembly, orchestrating arteriovenous differentiation and boundary formation. Despite these clearly defined rate-limiting roles of the EphB/ephrinB system for developmental angiogenesis, the mechanisms of the functions of EphB receptors and ephrinB ligands in the cells of the vascular system are poorly understood. Moreover, little evidence can be found in the recent literature regarding complementary EphB and ephrinB expression patterns that occur in the vascular system and that may bring cells into juxtapositional contact to allow bi-directional signaling between neighboring cells. This review summarizes the current knowledge of the role of EphB receptors and ephrinB ligands during embryonic vascular assembly and discusses recent findings on EphB/ephrinB-mediated cellular functions pointing to the crucial role of the Eph/ephrin system in controlling vascular homeostasis in the adult.Eph/ephrin work in the laboratory of the authors is supported by a grant from the Deutsche Forschungsgemeinschaft (Au83/3–2 within the SPP1069 "Angiogenesis")     

Distinctive Structure of the EphA3/Ephrin-A5 Complex Reveals a Dual Mode of Eph Receptor Interaction for Ephrin-A5

The Eph receptor tyrosine kinase/ephrin ligand system regulates a wide spectrum of physiological processes, while its dysregulation has been implicated in cancer progression. The human EphA3 receptor is widely upregulated in the tumor microenvironment and is highly expressed in some types of cancer cells. Furthermore, EphA3 is among the most highly mutated genes in lung cancer and it is also frequently mutated in other cancers. We report the structure of the ligand-binding domain of the EphA3 receptor in complex with its preferred ligand, ephrin-A5. The structure of the complex reveals a pronounced tilt of the ephrin-A5 ligand compared to its orientation when bound to the EphA2 and EphB2 receptors and similar to its orientation when bound to EphA4. This tilt brings an additional area of ephrin-A5 into contact with regions of EphA3 outside the ephrin-binding pocket thereby enlarging the size of the interface, which is consistent with the high binding affinity of ephrin-A5 for EphA3. This large variation in the tilt of ephrin-A5 bound to different Eph receptors has not been previously observed for other ephrins.     

High-content analysis of proapoptotic EphA4 dependence receptor functions using small-molecule libraries

Small-molecule compounds (SMCs) can provide an inexpensive and selective approach to modifying biological responses. High-content analysis (HCA) of SMC libraries can help identify candidate molecules that inhibit or activate cellular responses. In particular, regulation of cell death has important implications for many pathological conditions. Dependence receptors are a new classification of proapoptotic membrane receptors that, unlike classic death receptors, initiate apoptotic signals in the absence of their ligands. EphA4 has recently been identified as a dependence receptor that may have important functions in conditions as disparate as cancer biology and CNS injury and disease. To screen potential candidate SMCs that inhibit or activate EphA4-induced cell death, HCA of an SMC library was performed using stable EphA4-expressing NIH 3T3 cells. Our results describe a high-content method for screening dependence receptor-signaling pathways and demonstrate that several candidate SMCs can inhibit EphA4-mediated cell death.     

EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish

The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.     

Cancer somatic mutations disrupt functions of the EphA3 receptor tyrosine kinase through multiple mechanisms

The Eph receptor tyrosine kinases make up an important family of signal transduction molecules that control many cellular processes, including cell adhesion and movement, cell shape, and cell growth. All of these are important aspects of cancer progression, but the relationship between Eph receptors and cancer is complex and not fully understood. Genetic screens of tumor specimens from cancer patients have revealed somatic mutations in many Eph receptors. The most highly mutated Eph receptor is EphA3, but its functional role in cancer is currently not well established. Here we show that many EphA3 mutations identified in lung, colorectal, and hepatocellular cancers, melanoma, and glioblastoma impair kinase activity or ephrin ligand binding and/or decrease the level of receptor cell surface localization. These results suggest that EphA3 has ephrin- and kinase-dependent tumor suppressing activities, which are disrupted by somatic cancer mutations.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

Mammary epithelial‐specific knockout of the ephrin‐B2 gene leads to precocious epithelial cell death at lactation

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor‐ligand interaction requires direct cell–cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy‐lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo‐alveolar structures. Accordingly, massive epithelial cell death and expression of involution‐specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.     

Parcellation of the thalamus into distinct nuclei reflects EphA expression and function

Intercellular signaling via the Eph receptor tyrosine kinases and their ligands, the ephrins, acts to shape many regions of the developing brain. One intriguing consequence of Eph signaling is the control of mixing between discrete cell populations in the developing hindbrain, contributing to the formation of segregated rhombomeres. Since the thalamus is also a parcellated structure comprised of discrete nuclei, might Eph signaling play a parallel role in cell segregation in this brain structure? Analyses of expression reveal that several Eph family members are expressed in the forming thalamus and that cells expressing particular receptors form cellular groupings as development proceeds. Specifically, expression of receptors EphA4 or EphA7 and ligand ephrin-A5 is localized to distinct thalamic domains. EphA4 and EphA7 are often coexpressed in regions of the forming thalamus, with each receptor marking discrete thalamic domains. In contrast, ephrin-A5 is expressed by a limited group of thalamic cells. Within the ventral thalamus, EphA4 is present broadly, occasionally overlapping with ephrin-A5 expression. EphA7 is more restricted in its expression and is largely nonoverlapping with ephrin-A5. In mutant mice lacking one or both receptors or ephrin-A5, the appearance of the venteroposterolateral (VPL) and venteroposteromedial (VPM) nuclear complex is altered compared to wild type mice. These in vivo results support a role for Eph family members in the definition of the thalamic nuclei. In parallel, in vitro analysis reveals a hierarchy of mixing among cells expressing ephrin-A5 with cells expressing EphA4 alone, EphA4 and EphA7 together, or EphA7 alone. Together, these data support a model in which EphA molecules promote the parcellation of discrete thalamic nuclei by limiting the extent of cell mixing.     

Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion

Eph receptor-ephrin signals are important for controlling repulsive and attractive cell movements during tissue patterning in embryonic development. However, the dynamic cellular responses to these signals at cell-cell contact sites are poorly understood. To examine these events we have used cell microinjection to express EphB4 and ephrinB2 in adjacent Swiss 3T3 fibroblasts and have studied the interaction of the injected cells using time-lapse microscopy. We show that Eph receptors are locally activated wherever neighbouring cells make contact. This triggers dynamic, Rac-regulated membrane ruffles at the Eph-ephrin contact sites. Subsequently, the receptor and ligand cells retract from one another, concomitantly with the endocytosis of the activated Eph receptors and their bound, full-length ephrinB ligands. Both the internalization of the receptor-ligand complexes and the subsequent cell retraction events are dependent on actin polymerization, which in turn is dependent on Rac signalling within the receptor-expressing cells. Similar events occur in primary human endothelial cells. Our findings suggest a novel mechanism for cell repulsion, in which the contact between Eph-expressing and ephrin-expressing cells is destabilized by the localized phagocytosis of the ligand-expressing cell plasma membrane by the receptor-expressing cell.     

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.     

Regulation of EphA 4 kinase activity is required for a subset of axon guidance decisions suggesting a key role for receptor clustering in Eph function

Signaling by receptor tyrosine kinases (RTKs) is mediated by their intrinsic kinase activity. Typically, kinase-activating mutations result in ligand-independent signaling and gain-of-function phenotypes. Like other RTKs, Ephs require kinase activity to signal, but signaling by Ephs in vitro also requires clustering by their membrane bound ephrin ligands. The relative importance of Eph kinase activity and clustering for in vivo functions is unknown. We find that knockin mice expressing a mutant form of EphA4 (EphA4(EE)), whose kinase is constitutively activated in the absence of ephrinB ligands, are deficient in the development of thalamocortical projections and some aspects of central pattern generator rhythmicity. Surprisingly, other functions of EphA4 were regulated normally by EphA4(EE), including midline axon guidance, hindlimb locomotion, in vitro growth cone collapse, and phosphorylation of ephexin1. These results suggest that signaling of Eph RTKs follows a multistep process of induced kinase activity and higher-order clustering different from RTKs responding to soluble ligands.     

Myosin 1b functions as an effector of EphB signaling to control cell repulsion

Eph receptors and their membrane-tethered ligands, the ephrins, have important functions in embryo morphogenesis and in adult tissue homeostasis. Eph/ephrin signaling is essential for cell segregation and cell repulsion. This process is accompanied by morphological changes and actin remodeling that drives cell segregation and tissue patterning. The actin cortex must be mechanically coupled to the plasma membrane to orchestrate the cell morphology changes. Here, we demonstrate that myosin 1b that can mechanically link the membrane to the actin cytoskeleton interacts with EphB2 receptors via its tail and is tyrosine phosphorylated on its tail in an EphB2-dependent manner. Myosin 1b regulates the redistribution of myosin II in actomyosin fibers and the formation of filopodia at the interface of ephrinB1 and EphB2 cells, which are two processes mediated by EphB2 signaling that contribute to cell repulsion. Together, our results provide the first evidence that a myosin 1 functions as an effector of EphB2/ephrinB signaling, controls cell morphology, and thereby cell repulsion.     

EphA receptors direct the differentiation of mammalian neural precursor cells through a mitogen-activated protein kinase-dependent pathway

Ephrins are cell surface-associated ligands for Eph receptor tyrosine kinases and are implicated in repulsive axon guidance and cell migration. EphA2, 3, and 4 receptors and one of their cognate ligands, ephrin-A2, are expressed by cells in the subventricular zone and ganglionic eminence of the embryonic day 14.5 telencephalon and by neural precursor cells in vitro. Activation of EphA receptors in dissociated neural precursor cells in vitro facilitates the commitment to neuronal fates. The majority of ephrin-A1-induced neurons is immunoreactive for tyrosine hydroxylase. Blocking the signal by the extracellular domain of EphA in forebrain slices results in a decrease in neurogenesis. Extracellular signal-regulated kinase is activated by the ligand binding to EphA receptors and is involved in the neurogenesis through EphA receptors. Rap1, but not Ras, is activated in response to ephrin-A1. Our results identify EphA receptors as positive regulators of the mitogen-activated protein kinase pathway that exerts neurogenesis of neural precursor cells from the developing central nervous system.     

Bone cell interactions through Eph/ephrin

Bones cannot properly form or be maintained without cell-cell interactions through ephrin ligands and Eph receptors. Cell culture analysis and evaluation of genetic mouse models and human diseases reveal various ephrins and Eph functions in the skeletal system. Migration, attachment and spreading of mesenchymal stem cells are regulated by ephrinB ligands and EphB receptors. ephrinB1 loss-of-function is associated with craniofrontonasal syndrome (CFNS) in humans and mice. In bone remodeling, ephrinB2 is postulated to act as a “coupling stimulator.” In that case, bidirectional signaling between osteoclastic ephrinB2 and osteoblastic EphB4 suppresses osteoclastic bone resorption and enhances osteoblastic bone formation, facilitating the transition between these two states. Parathyroid hormone (PTH) induces ephrinB2 in osteoblasts and enhances osteoblastic bone formation. In contrast to ephrinB2, ephrinA2 acts as a “coupling inhibitor,” since ephrinA2 reverse signaling into osteoclasts enhances osteoclastogenesis and EphA2 forward signaling into osteoblasts suppresses osteoblastic bone formation and mineralization. Furthermore, ephrins and Ephs likely modulate pathological conditions such as osteoarthritis, rheumatoid arthritis, multiple myeloma and osteosarcoma. This review focuses on ephrin/Eph-mediated cell-cell interactions in bone biology.     

Structural and biophysical characterization of the EphB4*ephrinB2 protein-protein interaction and receptor specificity

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.