Structure and Thermodynamic Characterization of the EphB4/Ephrin-B2 Antagonist Peptide Complex Reveals the Determinants for Receptor Specificity

Chrencik, Jill E.; Brooun, Alexei; Recht, Michael I.; Kraus, Michelle L.; Koolpe, Mitchell; Kolatkar, Anand; Bruce, Richard H.; Martiny-Baron, Georg; Widmer, Hans; Pasquale, Elena B.; Kühn, Peter

doi:10.1016/j.str.2005.11.011

Cited by 83 publications

(104 citation statements)

References 44 publications

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“…Protein Expression and Purification-The human EphB4 receptor was expressed and purified in insect cells as described elsewhere (19). The wild type EphB4 construct was used as a template for the generation of site-specific mutants.…”

Section: Methodsmentioning

confidence: 99%

“…All experiments were performed with a Microcal MCS ITC at 25°C. Titrations were completed as described (19). 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.…”

Section: Methodsmentioning

confidence: 99%

“…Crystal structures have been previously determined for ephrin ligands (ephrinB2, ephrinB1) and Eph receptors (EphB2, EphB4) as well as receptor-ligand complexes (EphB2⅐ephrinB2, EphB2⅐ephrinA5, EphB4-TNYL-RAW (8,10,19). The crystal structure of the homodimeric ephrinB2 ligand reveals a ␤-barrel structure arranged in a deviation of the common Greek Key topology (17).…”

mentioning

confidence: 99%

“…This critical loop shares high sequence homology in both A and B subclasses, respectively, and is involved in receptor-ligand heterodimerization as well as in ephrinB2 homodimerization (10,17). In addition, the crystal structures of the ligand binding domains of EphB2 and EphB4, which share only 44% sequence identity, show a similar jellyroll folding topology, with 13 antiparallel ␤-sheets connected by loops of varying length (18,19). The crystal structure of the EphB4 receptor in complex with the phage display derived TNYL-RAW peptide revealed that the peptide binds to the ephrin binding cavity of the receptor, effectively inhibiting interaction with the ligand (19).…”

mentioning

confidence: 99%

“…In addition, the crystal structures of the ligand binding domains of EphB2 and EphB4, which share only 44% sequence identity, show a similar jellyroll folding topology, with 13 antiparallel ␤-sheets connected by loops of varying length (18,19). The crystal structure of the EphB4 receptor in complex with the phage display derived TNYL-RAW peptide revealed that the peptide binds to the ephrin binding cavity of the receptor, effectively inhibiting interaction with the ligand (19). Furthermore, the structure of the EphB2⅐ephrinB2 complex showed that the ligand binding channel of the receptor is located at the upper convex surface of EphB2 and is formed by the flexible J-K, G-H, and D-E loops, which become ordered to accommodate the solvent-exposed ephrin G-H loop (10).…”

mentioning

confidence: 99%

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

Chrencik

Brooun

Kraus

et al. 2006

Journal of Biological Chemistry

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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...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Chrencik

Brooun

Kraus

et al. 2006

Journal of Biological Chemistry

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Ephrin B2/EphB4 Mediates the Actions of IGF-I Signaling in Regulating Endochondral Bone Formation

Wang

Menendez

Fong

et al. 2014

Journal of Bone and Mineral Research

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Ephrin B2/EphB4 mediates interactions among osteoblasts (OB), osteoclasts (OCL), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of IGF1 and PTH on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs and osteocytes, and the expression of EphB4 in OBs and osteocytes were dramatically decreased in global IGF-I knockout mice. Inactivation of EphB4 by EphB4 siRNA in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF-I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB-OCL co-cultures with the EphB4 specific peptide TNYL-RAW or deletion of ephrin B2 in OCL prior to co-culture led to fewer and smaller TRAP positive cells, decreased expression of OB differentiation markers, and blunted response to IGF-I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF-I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL-RAW significantly decreased both basal and IGF1-induced expression of type II and type X collagen. In the co-cultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL-RAW decreased the numbers of TRAP positive cells and the expression of NFATc1 and RANK, and blocked their stimulation by IGF-I. Our data indicate that IGF-I/IGF-IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell-cell communication.

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EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

Chen¹,

Bai

Liu

et al. 2015

J of Cellular Biochemistry

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Eph receptor (Eph)-ephrin signaling plays an important role in organ development and tissue regeneration. Bidirectional signaling of EphB4– ephrinB2 regulates cardiovascular development. To assess the role of EphB4–ephrinB2 signaling in cardiac lineage development, we utilized two GFP reporter systems in embryonic stem (ES) cells, in which the GFP transgenes were expressed in Nkx2.5+ cardiac progenitor cells and in α-MHC+ cardiomyocytes, respectively. We found that both EphB4 and ephrinB2 were expressed in Nkx2.5-GFP+ cardiac progenitor cells, but not in α-MHC-GFP+ cardiomyocytes during cardiac lineage differentiation of ES cells. An antagonist of EphB4, TNYL-RAW peptides, that block the binding of EphB4 and ephrinB2, impaired cardiac lineage development in ES cells. Inhibition of EphB4–ephrinB2 signaling at different time points during ES cell differentiation demonstrated that the interaction of EphB4 and ephrinB2 was required for the early stage of cardiac lineage development. Forced expression of human full-length EphB4 or intracellular domain-truncated EphB4 in EphB4-null ES cells was established to investigate the role of EphB4-forward signaling in ES cells. Interestingly, while full-length EphB4 was able to restore the cardiac lineage development in EphB4-null ES cells, the truncated EphB4 that lacks the intracellular domain of tyrosine kinase and PDZ motif failed to rescue the defect of cardiomyocyte development, suggesting that EphB4 intracellular domain is essential for the development of cardiomyocytes. Our study provides evidence that receptor-kinase-dependent EphB4-forward signaling plays a crucial role in the development of cardiac progenitor cells.

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Structure and Thermodynamic Characterization of the EphB4/Ephrin-B2 Antagonist Peptide Complex Reveals the Determinants for Receptor Specificity

Cited by 83 publications

References 44 publications

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

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

Ephrin B2/EphB4 Mediates the Actions of IGF-I Signaling in Regulating Endochondral Bone Formation

EphB4 Forward‐Signaling Regulates Cardiac Progenitor Development in Mouse ES Cells

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