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 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. 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. The protein-protein interaction between the membrane-bound Eph receptor tyrosine kinases with the membrane-bound ephrin ligands have now been reported in the overexpression/dys-regulation in numerous tumor cell lines (1Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Crossref PubMed Scopus (257) Google Scholar). 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 (2Pasquale E.B. Nat. Rev. Mol. Cell Biol. 2005; 6: 462-475Crossref PubMed Scopus (839) Google Scholar, 3Wilkinson D.G. Nat. Rev. Neurosci. 2001; 2: 155-164Crossref PubMed Scopus (518) Google Scholar, 4Wilkinson D.G. Int. Rev. Cytol. 2000; 196: 177-244Crossref PubMed Google Scholar). 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 (5Blits-Huizinga C.T. Nelersa C.M. Malhotra A. Liebl D.J. IUBMB Life. 2004; 56: 257-265Crossref PubMed Scopus (51) Google Scholar). Understanding the EphB4·ephrinB2 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) (12Holland S.J. Gale N.W. Mbamalu G. Yancopoulos G.D. Henkemeyer M. Pawson T. Nature. 1996; 383: 722-725Crossref PubMed Scopus (455) Google Scholar, 13Schmucker D. Zipursky S.L. Cell. 2001; 105: 701-704Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Therefore, a cellular response is conducted only upon cell-cell contact. The Eph receptors are divided into two subclasses, A and B, based on both sequence conservation and binding preferences with the ephrins (6Gale N.W. Holland S.J. Valenzuela D.M. Flenniken A. Pan L. Ryan T.E. Henkemeyer M. Strebhardt K. Hirai H. Wilkinson D.G. Pawson T. Davis S. Yancopoulos G.D. Neuron. 1996; 17: 9-19Abstract Full Text Full Text PDF PubMed Scopus (748) Google Scholar). The receptors have a modular domain architecture, extracellularly characterized by an N-terminal ligand binding domain, a cysteine-rich domain, and two fibronectin type III-like repeats. Intracellularly, the receptors consist of a juxtamembrane domain, a conserved tyrosine kinase domain, a C-terminal sterile α-domain, and a PDZ binding motif. The ephrin ligands are also divided into two subclasses, A and B; ephrinA ligands are anchored to the membrane through a glycosylphosphatidylinositol linker, whereas members of the B-subclass are tethered to the membrane by a transmembrane region and contain a cytoplasmic PDZ domain binding motif. In general, EphA receptors (EphA1-EphA10) are promiscuously activated by ephrinA ligands (ephrinA1-ephrinA6), whereas EphB receptors (EphB1-EphB6) are promiscuously activated by ephrinB ligands (ephrinB1-ephrinB3) (7Committee E.N. Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar). Cross-subclass interactions have been identified specifically between EphB2 and ephrinA5 (8Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (365) Google Scholar) and between EphA4 and ephrinB2/B3 (9Takemoto M. Fukuda T. Sonoda R. Murakami F. Tanaka H. Yamamoto N. Eur. J. Neurosci. 2002; 16: 1168-1172Crossref PubMed Scopus (38) Google Scholar); however, these interactions are rare and appear to be exceptions to the general rule. Unlike the other promiscuous receptors in this family, however, the EphB4 receptor has a distinctive specificity for a single ligand, ephrinB2 while binding only weakly to both ephrinB1 and ephrinB3. EphrinB2, on the other hand, binds to several receptors within the B-subclass (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar). The ligand specificity of the EphB4 receptor is somewhat surprising, since the receptor has the characteristics of a promiscuous receptor, including a large and hydrophobic binding cavity and a flexible “lid” capable of accommodating a range of ligands (11Hopkins A.L. Mason J.S. Overington J.P. Curr. Opin. Struct. Biol. 2006; 16: 127-136Crossref PubMed Scopus (402) Google Scholar). Here we provide a structural perspective describing the molecular determinants of EphB4 specificity. Crystal structures have been previously determined for ephrin ligands (ephrinB2, ephrinB1) and Eph receptors (EphB2, EphB4) as well as receptor-ligand complexes (EphB2·ephrinB2, EphB2·ephrinA5, EphB4-TNYL-RAW (8Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (365) Google Scholar, 10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, 19Chrencik J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The crystal structure of the homodimeric ephrinB2 ligand reveals a β-barrel structure arranged in a deviation of the common Greek Key topology (17Toth J. Cutforth T. Gelinas A.D. Bethoney K.A. Bard J. Harrison C.J. Dev. Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Comparison of the crystal structures of both apo and receptor-bound ephrinB2 revealed that the and high affinity is conserved in position between the two This critical high sequence in both A and subclasses, and is in receptor-ligand as well as in ephrinB2 (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, J. Cutforth T. Gelinas A.D. Bethoney K.A. Bard J. Harrison C.J. Dev. Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the crystal structures of the ligand binding of EphB2 and which only sequence a with by of J.P. Henkemeyer M. Nikolov D.B. Nature. PubMed Scopus Google Scholar, 19Chrencik J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The crystal structure of the EphB4 receptor in complex with the peptide revealed that the peptide binds to the ephrin binding cavity of the receptor, inhibiting interaction with the ligand J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). the structure of the complex that the ligand binding of the receptor is at the of EphB2 and is by the flexible and which to the ephrin (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar). A affinity also identified at the of the receptor which with the of the that the EphB4·ephrinB2 interaction is important in angiogenesis and that EphB4 is in several tumor (1Dodelet V.C. Pasquale E.B. Oncogene. 2000; 19: 5614-5619Crossref PubMed Scopus (257) Google Scholar, M. A.D. 2002; PubMed Scopus Google Scholar, N. F. 2002; PubMed Scopus Google Scholar, G. L. A. 14: Full Text Full Text PDF PubMed Scopus Google modulating this protein-protein interaction is a to tumor angiogenesis and tumor In of high EphB4 with and tumor G. D. A. Oncogene. Google Scholar, G. A. J. Cell PubMed Google Scholar, N. R. S. G. S. A. J. Cell 2002; PubMed Google Scholar). EphB4 is also in and is to G. D. A. 1996; PubMed Scopus Google Scholar). is evidence that the EphB4 cell and that ephrinB2-expressing with the EphB4 to angiogenesis and tumor a EphB4 mutant has been to cancer cell that kinase is not an for at in cancer M. A.L. Koolpe M. Pasquale E.B. S. A. 2004; PubMed Scopus Google Scholar). have more that the extracellular domain of EphB4 is a therapeutic the extracellular domain of EphB4 to both and in an of tumor in N. R. L. S. 2006; PubMed Scopus Google Scholar, G. T. F. N. S. D. 2004; 6: PubMed Google Scholar). have identified a peptide that the EphB4·ephrinB2 interaction in the high range M. R. M. Pasquale E.B. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). a more understanding of the role of EphB4·ephrinB2 in and in forms of pathological angiogenesis is now To therapeutic development and EphB4 we determined the crystal structure of the EphB4·ephrinB2 complex to high In we conducted and to the role of several within the ligand binding cavity of EphB4 in to the binding of both ephrinB2 and the antagonistic peptide. These results will the development of for of small molecule compounds for as and to the biology of EphB4·ephrinB2 and EphB4 receptor and in as J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The type EphB4 as a for the of The ephrinB2 domain, designed based on the previously structure and into a a peptide. the large of ephrinB2 on a at a of of for in of of ephrinB2 protein and a The ligand by affinity and with by affinity to the and an N-terminal the The EphB4·ephrinB2 complex with a of ephrinB2 at in and The complex by affinity by to The of the complex and EphB4·ephrinB2 complex to in a and and by at a of and EphB4·ephrinB2 in and in the group and one of receptor and one of ligand in the at the on and in A. Scholar). The structure by molecular with the structure as a (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, Biol. PubMed Scopus Google Scholar). The structure by a by in and with Biol. PubMed Scopus Google Scholar, Cowan M. A. PubMed Scopus Google Scholar). The structure with Isothermal and ligands into and in titration calorimetry titration experiments with a at as J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). EphB4 type or in the cell at a of and the either ephrinB2 or of with the mutant of EphB4 with in the and EphB4 in the for these a of and at at the peptide as J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, J. PubMed Scopus Google Scholar). peptide by A of EphB4 in peptide as a in the and a the measurements by in the of EphB4 with of peptide in the of in the and of as a for The to for at and measurements with a and and by the a binding The for at protein and of the response is and ephrinB2 to and by a of and at The structure to with an of and a of Unlike of the of the which of a of the EphB4·ephrinB2 complex consist of a of for a range whereas that the complex a at in the range (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar). analysis of the EphB4·ephrinB2 complex in a range to that the EphB4·ephrinB2 complex as a Brooun and J. in The structure of the EphB4·ephrinB2 complex is to that of the with a deviation of deviation is however, the structure of the receptor with the EphB4-TNYL-RAW and with of and in the The ephrinB2 ligand between previously apo and receptor-bound only and (10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, J. Cutforth T. Gelinas A.D. Bethoney K.A. Bard J. Harrison C.J. Dev. Cell. 2001; 1: 83-92Abstract Full Text Full Text PDF PubMed Scopus (85) Google for the in is for the cell is the and is the of of that of and are the and structure for of the not at of structural of The in is for the is the and is the of of that and are the and structure for of the not at of structural in a high affinity EphB4·ephrinB2 is by of the ligand into the and hydrophobic of the EphB4 receptor, receptor and M. receptor-ligand binding in this and in interactions with receptor the receptor binding cavity in the and forms hydrophobic interactions with and whereas with the receptor to the of the receptor in between the and in hydrophobic interactions with and In to all crystal its position by in a interaction with the receptor are at the receptor-ligand forms a with as well as a with whereas forms a with the of to the of the ephrinB2 with and with and which are both of the high affinity ligand sequence The of this interaction at the EphB4·ephrinB2 is to to the high affinity of this receptor-ligand of at at for the EphB4·ephrinB2 EphB4 is in and ephrinB2 is in of the in a novel position with to previously structures in to with to the a of the high affinity to the ligand binding cavity, by ligand and and receptor and D. This region of the complex the corresponding with a deviation of and is characterized by and In interactions between in and in and provide the binding of this interactions between and and between and the binding in this the of the EphB4·ephrinB2 interaction is in with that previously in the within the receptor that the ligand binding The EphB4 a position with previously crystal structures and is the ligand and the The corresponding the on the other hand, is only the a more binding In the in position by to between the two complex the in structure also in position by to between the EphB4-TNYL-RAW with the apo of EphB4 to of the of the and A unique to EphB4 is a in the which is in all other Eph has been that this to the ligand binding specificity to the EphB4 receptor D.B. Li C. Barton W.A. J.P. 2005; PubMed Scopus Google Scholar). Indeed, two of the the of the and with the forms a hydrophobic with whereas a to with In the and which two of the ligand binding also to the ligand. The is by between the EphB4 and ephrinB2 whereas the only by between the two EphB4 comparison and structural analysis of the EphB4 and EphB2 receptors suggested that one in EphB4 is particularly important in the specificity of the EphB4·ephrinB2 The corresponding in is conserved both A and and only in the EphB4 in with the high affinity ephrin including and and is in to a critical for receptor of an at position in the EphB4·ephrinB2 structure that a result between the arginine and The corresponding on the other hand, is to a interaction with to its position within the ligand binding also with the the peptide in the EphB4-TNYL-RAW structure J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google whereas the forms with the peptide. the conserved is in position by as with complex structures (8Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (365) Google Scholar, 10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, 19Chrencik J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar) and is within the hydrophobic of the receptor, its position in the complex is the In the position of the of the EphB2 receptor to the ligand and the receptor to with the ephrinB2 The with the of EphB4 to a novel position the ligand of EphB4 of by the EphB4 and to the corresponding found in EphB2 The EphB4 based on their binding to peptide. analysis the that Leu-95 is a critical for binding of the peptide the mutant not binding of the peptide in EphB4 and an in binding affinity of the of EphB4 on binding to is not to is not to in a on the structural information and binding by two EphB4 and for a more thermodynamic analysis of their binding to both ephrinB2 and the peptide ligand reported EphB4 binds to ephrinB2 with an affinity of and a of J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). of EphB4 to has on the binding affinity or of ephrinB2 binding In of EphB4 Leu-95 to the binding affinity of ephrinB2 by of of ephrinB2 to all forms of EphB4 is and the binding of ephrinB2 is more with the in EphB4 for type experiments in a with of a to that reported binding of ephrinB2 to EphB4 in a of in with the of in that is not to ephrinB2 binding the of the of peptide and ephrinB2 to the EphB4 ephrin binding domain and EphB4 in a of the peptide to the and forms of EphB4 also by binds to EphB4 with an affinity of and a of J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In to the of in EphB4 on the interaction of EphB4 with ephrinB2, of EphB4 of either to or Leu-95 to the affinity of the EphB4-TNYL-RAW interaction and of the peptide to all forms of EphB4 is characterized by an thermodynamic analysis reveals that binding to the EphB4 ligand binding domain is an enthalpically driven process, whereas ephrinB2 binding to EphB4 is an entropically driven process. The in the binding are with the structural of the hydrophobic ligand within the hydrophobic receptor binding entropically the interaction through the of the In the ephrinB2 ligand is both in apo and receptor-bound The small of the by the flexible receptor and Unlike ephrinB2, however, the peptide ligand will of upon EphB4 binding, in an This is by an to the of both and at the A thermodynamic analysis is however, to to apo EphB4. be that we the ephrinB2 extracellular domain in in a whereas the ephrins for crystal structure in not A conserved in ephrinB2 at in to the affinity with its is the of the and its the of the the not in is that a at this with the of receptor-ligand in the crystal have suggested that more a an role in J.P. Nikolov D.B. Biol. 2002; PubMed Scopus Google Scholar). We have determined the crystal structure of the EphB4 receptor ligand binding domain in complex with the extracellular domain of its ligand, ephrinB2, and identified the determinants for EphB4 specificity. A sequence with members of the EphB reveals that the EphB4 receptor a conserved arginine and a at position A previously to result in with the antagonistic peptide ligand J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Here we that this also result in with in the of ephrinB2 to the unique of the of EphB4. A of an arginine at position of the EphB4 receptor to be to a structural of the receptor is the novel position of the conserved in the high affinity sequence of the ephrinB2 ephrinB2 is conserved in structure in both receptor-bound and apo to be within the to to a and sequence with ephrinB1 and EphB4 binds only weakly to both of these ligands while high affinity for ephrinB2. the B-subclass ephrin is interesting to on EphB4 binds ephrinB2 other B-subclass sequence with the high affinity ephrinB2 at position which is a in ephrinB1 and a in ephrinB2. not interactions with the small of the within the receptor binding cavity and the hydrophobic of the binding of a tyrosine on the ephrinB2 structure the of the EphB4 to the for the affinity of EphB4 for The is also to the ephrinB2 but in the which a tyrosine of the forms critical interactions with the EphB binding cavity in the complex crystal structures (8Himanen J.P. Chumley M.J. Lackmann M. Li C. Barton W.A. Jeffrey P.D. Vearing C. Geleick D. Feldheim D.A. Boyd A.W. Henkemeyer M. Nikolov D.B. Nat. Neurosci. 2004; 7: 501-509Crossref PubMed Scopus (365) Google Scholar, 10Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (270) Google Scholar, 19Chrencik J.E. Brooun A. Recht M.I. Kraus M.L. Koolpe M. Kolatkar A.R. Bruce R.H. Martiny-Baron G. Widmer H. Pasquale E.B. Kuhn P. Structure. 2006; 14: 321-330Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). In the crystal structures to the of the binding cavity, to the receptor of a tyrosine on the structure not the of the ligand binding cavity, and this with several on the of the in structure the of ephrinB2 is in a novel within the hydrophobic binding and interactions with and the of a tyrosine at this position result in both within the receptor binding cavity and a of the with the affinity of EphB4 for into the of the crystal EphB4 and ephrinB2 only both in the and in this is to the structure the more that of the ligand binding domain, which is with Vearing C. Lackmann M. H. J. K. A. Nikolov D. Boyd A.W. J. Biol. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, To C. J.P. Nikolov D.B. Boyd A.W. Lackmann M. J. Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). have that complexes are on the high affinity the and a in the cysteine-rich which is to the ligand binding domain and previously reported to receptor-ligand affinity The thermodynamic between peptide and ephrin binding be in the of to to the Eph receptor of will an understanding of the and of small molecule The ephrin ligand, with entropically driven binding, with members of the EphB In the with enthalpically driven binding, is a of the EphB4·ephrinB2 The of the enthalpically driven peptide binding is with the by the group of the selectivity of binding of H. S. A. A. S. Int. J. Cell Biol. 2004; PubMed Scopus Google Scholar). are to the between the specificity of the interaction and the of the to the binding is a or a more general of the interaction of We have characterized the interaction between the EphB4 ligand binding domain and the extracellular domain of ephrinB2 both and We now the structural determinants that confer the high affinity and specificity of EphB4 for ephrinB2 and the role of the critical EphB4 of EphB4 and ephrinB2 will provide more for understanding the of by their The of in EphB4 ligand binding the to and ligand results with the information other Eph complex structures the to small molecule compounds and with that the EphB4·ephrinB2 We Pasquale and C. for and We also the of the at the for in The and is by of and