Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity

Abstract

EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln12 and Glu14 and ephrin-B2. Mutation of Gln12 and Glu14 does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ∼10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln12 and Glu14 with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes. EphA and EphB receptors preferentially bind ephrin-A and ephrin-B ligands, respectively, but EphA4 is exceptional for its ability to bind all ephrins. Here, we report the crystal structure of the EphA4 ligand-binding domain in complex with ephrin-B2, which represents the first structure of an EphA-ephrin-B interclass complex. A loose fit of the ephrin-B2 G-H loop in the EphA4 ligand-binding channel is consistent with a relatively weak binding affinity. Additional surface contacts also exist between EphA4 residues Gln12 and Glu14 and ephrin-B2. Mutation of Gln12 and Glu14 does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ∼10-fold reduced affinity for ephrin-B ligands, indicating that the surface contacts are critical for interclass but not intraclass ephrin binding. Thus, EphA4 uses different strategies to bind ephrin-A or ephrin-B ligands and achieve binding promiscuity. NMR characterization also suggests that the contacts of Gln12 and Glu14 with ephrin-B2 induce dynamic changes throughout the whole EphA4 ligand-binding domain. Our findings shed light on the distinctive features that enable the remarkable ligand binding promiscuity of EphA4 and suggest that diverse strategies are needed to effectively disrupt different Eph-ephrin complexes. IntroductionThe Eph receptors represent the largest family of tyrosine kinases, with 16 members divided into two classes, EphA and EphB. This subdivision is based on sequence conservation and binding preferences for their ligands, the ephrins, which are also divided into A and B classes. There are 10 EphA and 6 EphB receptors in mammals and chick, which can bind to six glycosylphosphatidylinositol-anchored ephrin-A ligands or three transmembrane ephrin-B ligands to mediate an extremely wide spectrum of biological responses through signals that are generated by both receptor and ligand activation (1Pasquale E.B. Nat. Rev. Mol. Cell Biol. 2005; 6: 462-475Crossref PubMed Scopus (845) Google Scholar, 2Pasquale E.B. Cell. 2008; 133: 38-52Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar).All of the Eph receptors share the same modular structure, which comprises a juxtamembrane region, a tyrosine kinase domain, a C-terminal sterile α-motif domain, and a PDZ-binding motif in the intracellular region. In the extracellular portion, there are an N-terminal ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats. The ephrin-binding domain is responsible for ligand recognition and is composed of 11 antiparallel β-strands organized in a jellyroll β-sandwich architecture, which is conserved among EphA and EphB receptors (3Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (91) Google Scholar, 4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Qin H. Shi J. Noberini R. Pasquale E.B. Song J. J. Biol. Chem. 2008; 283: 29473-29484Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). The ectodomain of the ephrins is also conserved and consists of an eight-stranded β-barrel with a Greek key topology, including several large and highly conserved functional loops, such as the G-H and C-D loops (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar, 9Toth 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 (86) Google Scholar), which are very flexible in solution (10Ran X. Qin H. Liu J. Fan J.S. Shi J. Song J. Proteins. 2008; 72: 1019-1029Crossref PubMed Scopus (16) Google Scholar).The formation of a complex between an Eph receptor and an ephrin is centered around the insertion of the solvent-exposed ephrin G-H loop into the Eph receptor hydrophobic channel formed by the convex sheet of four β-strands together with the D-E, J-K, and G-H loops. These interactions are mostly hydrophobic and, together with an adjacent mostly polar surface region, form the high affinity interface of Eph receptor-ephrin complexes, which is involved in receptor-ephrin dimerization (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). Other interfaces contribute to Eph-ephrin binding, including (i) additional residues on both the receptor and ephrin surfaces; (ii) a low affinity interface also involving the Eph receptor ligand-binding domain, which was identified in the EphB2-ephrin-B2 complex and appears to mediate the association of two receptor-ephrin dimers (tetramerization) (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar), and (iii) an interface involving the cysteine-rich region adjacent to the Eph receptor ligand-binding domain, which was identified by mutagenesis in the EphA3-ephrin-A5 complex but has not been structurally characterized and which might be implicated in higher order clustering (11Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar).Although Eph receptors interact promiscuously with ephrins of the same class, they rarely interact with ephrins of the other class. A variety of factors appear to contribute to class specificity. B class Eph-ephrin interactions require considerable structural rearrangements of both the receptor and the ephrin, whereas EphA receptors and ephrin-A ligands appear to undergo smaller rearrangements when forming a complex (8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). Differences in critical residues located in the interacting and sequence in the class loop of the Eph receptors also to a in class (3Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (91) Google Scholar, 4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Qin H. Shi J. Noberini R. Pasquale E.B. Song J. J. Biol. Chem. 2008; 283: 29473-29484Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). However, of interclass binding also can bind and EphA4 can bind all three ephrin-B ligands E.B. Nat. Neurosci. 2004; 7: PubMed Scopus Google Scholar).The structure of the ephrin-binding domain in complex with has been 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 (368) Google and that the two interact through the high affinity which is reduced with intraclass complexes, in a binding affinity. EphA4 binding to ephrin-B ligands is also to ephrin-A ligands. However, the of interclass interactions has been in biological EphA4 with formation H. P. 2005; PubMed Scopus Google Scholar), whereas EphA4 with ephrin-B2 in the and of the A. K. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, Nature. PubMed Scopus Google Scholar), A. C. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and formation in the K. M. Boyd A.W. R. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. J. R. Dev. 2001; PubMed Scopus Google Scholar, M.I. Cowan C.A. P. P. Henkemeyer M. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J.M. A. R. PubMed Scopus Google Scholar), and of by M.I. Henkemeyer M. 2005; PubMed Scopus Google Scholar).The distinctive ability of EphA4 to bind both ephrin-A and ephrin-B ligands an to the structural the promiscuity of Eph receptor-ephrin but structural has been for complexes. In we report the crystal structure of the complex and a polar region, structurally the ephrin-binding as critical for binding. also characterized the complex in solution by NMR which represents the first NMR of an Eph-ephrin complex. that EphA4 uses different strategies for binding ephrin-A ephrin-B ligands, remarkable binding promiscuity between Eph receptors and ephrin ligands appears to be a key Eph-ephrin to a wide of biological the structural promiscuity of Eph receptor-ephrin binding is for the the biological of the Eph and is also critical for the of to Eph-ephrin EphA4 is the Eph receptor that can bind with affinity all ephrin-A and ephrin-B ligands E.B. Nat. Neurosci. 2004; 7: PubMed Scopus Google Scholar). Here, we the structure of the which represents the first structure of a complex between an EphA receptor and an ephrin-B of the complex is very to for other Eph-ephrin complexes. The high affinity interface of the complex can be divided into two relatively mostly the hydrophobic interactions between the EphA4 ligand-binding channel and the ephrin-B2 G-H The which was in (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google but was reduced in the complex and in the complex 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 (368) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar), polar interactions between surface residues Gln12 and Glu14 in EphA4 and and in ephrin-B2. not for a affinity binding interface that mediate of two to that for the EphB2-ephrin-B2 complex (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar). an interface is also not in the and 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google structural are in EphA4 complex formation with ephrin-B2, including in the and β-strands as as the D-E, and loops. structural rearrangements in also been for EphB receptors ephrin binding (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, Y. Nikolov D. Himanen J.P. Biol. 2009; PubMed Scopus Google Scholar). In structural changes been formation of the complex (8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar).The structure also the relatively weak affinity of the interclass binding with intraclass such as of with ephrin-B2 and with ephrin-B2 of sequence a hydrophobic in the EphB2-ephrin-B2 complex is in the complex. a of the EphA4 loop and does not contacts with the ephrin-B2 G-H loop that the structurally loop region is in the complex with and that the binding affinity of for is 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 (368) Google Scholar), in for the low binding affinity between EphA4 and ephrin-B2. However, for and mutant that the surface contacts formed between EphA4 residues Gln12 and Glu14 and ephrin-B2 residues and by ∼10-fold the binding affinity and a critical in interclass of the two EphA4 residues involved in the interface an EphA4 mutant that does not appear to undergo structural changes or in the ephrin-binding This is the binding affinity of the EphA4 mutant for two that bind the the polar surface region of EphA4 does not appear to be for the intraclass binding with ephrin-A ligands, which a fit between the EphA4 ligand-binding channel and the ephrin-A G-H This is the for the the surface region is extremely reduced (8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). region is not or in the other and of ephrin-B2 been to also interact with residues on the surface of EphB receptors (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), which the in the affinity of mutant ephrin-B2 for EphB However, we that the in binding be to the changes in the of the mutant ephrin by the in the binding of mutant ephrin-B2 to EphA4 to EphB receptors suggests that the surface region is critical for the interclass binding the intraclass binding. the to ephrin-B2 is a in and be involved in the with EphA4 Gln12 that is in the complex. However, the binding affinity of the EphA4 mutant for ephrin-B2 or is reduced with that the between ephrin-B2 and EphA4 might a in interclass binding the between ephrin-B2 and EphA4 characterization also the first into the dynamic with binding in which not be of the complex. the NMR that EphA4 residues ephrin-B2 binding to that or are located the high affinity binding residues in the EphA4 and are they are the binding in the crystal structure, residues not appear to undergo changes ephrin-B2 binding In the interactions by EphA4 Gln12 and Glu14 are the residues by the binding of ephrin-B2 are to the EphA4 interface in with ephrin-B2. Thus, contacts by Gln12 Glu14 the EphA4 ligand-binding domain. is to that involving and dynamic changes in EphA4 that are by ephrin-B binding and are critical for biological However, we not for be that a very weak might exist for to form high order such as in to is with EphB2-ephrin-B2 (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar). be to in NMR as J. R. J. Chem. B. 2008; PubMed Scopus Google Scholar).The complex structure that we together with for other Eph receptor-ephrin a in the of the two of the high affinity interface to intraclass or interclass Eph receptor-ephrin binding. appears that intraclass binding is or by the hydrophobic Eph G-H loop region of the the fit of the ephrin G-H loop into the Eph channel is for the A the B class. interclass binding on the channel region of the the loose interclass fit of the EphB channel and the ephrin-A G-H loop in the binding affinity among the structurally characterized we that interclass binding uses a the very weak binding through the Eph G-H loop is by interactions in the polar surface region of the In EphA4 the promiscuity among the Eph a of in Eph receptor-ephrin the of to Eph-ephrin interactions be and diverse strategies be needed on the Eph receptor and the ephrin with we that two that the ephrin-binding channel of EphA4 the binding of ephrins but not we not of ephrin-B2 and binding to EphA4 that the binding of other ephrins R. M. R. Y. Pasquale E.B. J. Biol. Chem. 2008; 283: Full Text Full Text PDF PubMed Scopus Google Scholar). The structural we and the of the EphA4 and ephrin-B2 also suggest that is to EphA4 binding to ephrin-B but not ephrin-A ligands by the polar surface region of the high affinity whereas the binding of ephrin-A ligands to EphA4 be by contacts in the channel region. strategies the biological of intraclass interclass binding and for the of EphA4 IntroductionThe Eph receptors represent the largest family of tyrosine kinases, with 16 members divided into two classes, EphA and EphB. This subdivision is based on sequence conservation and binding preferences for their ligands, the ephrins, which are also divided into A and B classes. There are 10 EphA and 6 EphB receptors in mammals and chick, which can bind to six glycosylphosphatidylinositol-anchored ephrin-A ligands or three transmembrane ephrin-B ligands to mediate an extremely wide spectrum of biological responses through signals that are generated by both receptor and ligand activation (1Pasquale E.B. Nat. Rev. Mol. Cell Biol. 2005; 6: 462-475Crossref PubMed Scopus (845) Google Scholar, 2Pasquale E.B. Cell. 2008; 133: 38-52Abstract Full Text Full Text PDF PubMed Scopus (963) Google Scholar).All of the Eph receptors share the same modular structure, which comprises a juxtamembrane region, a tyrosine kinase domain, a C-terminal sterile α-motif domain, and a PDZ-binding motif in the intracellular region. In the extracellular portion, there are an N-terminal ligand-binding domain, a cysteine-rich region, and two fibronectin type III repeats. The ephrin-binding domain is responsible for ligand recognition and is composed of 11 antiparallel β-strands organized in a jellyroll β-sandwich architecture, which is conserved among EphA and EphB receptors (3Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (91) Google Scholar, 4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Qin H. Shi J. Noberini R. Pasquale E.B. Song J. J. Biol. Chem. 2008; 283: 29473-29484Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). The ectodomain of the ephrins is also conserved and consists of an eight-stranded β-barrel with a Greek key topology, including several large and highly conserved functional loops, such as the G-H and C-D loops (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar, 9Toth 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 (86) Google Scholar), which are very flexible in solution (10Ran X. Qin H. Liu J. Fan J.S. Shi J. Song J. Proteins. 2008; 72: 1019-1029Crossref PubMed Scopus (16) Google Scholar).The formation of a complex between an Eph receptor and an ephrin is centered around the insertion of the solvent-exposed ephrin G-H loop into the Eph receptor hydrophobic channel formed by the convex sheet of four β-strands together with the D-E, J-K, and G-H loops. These interactions are mostly hydrophobic and, together with an adjacent mostly polar surface region, form the high affinity interface of Eph receptor-ephrin complexes, which is involved in receptor-ephrin dimerization (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). Other interfaces contribute to Eph-ephrin binding, including (i) additional residues on both the receptor and ephrin surfaces; (ii) a low affinity interface also involving the Eph receptor ligand-binding domain, which was identified in the EphB2-ephrin-B2 complex and appears to mediate the association of two receptor-ephrin dimers (tetramerization) (4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar), and (iii) an interface involving the cysteine-rich region adjacent to the Eph receptor ligand-binding domain, which was identified by mutagenesis in the EphA3-ephrin-A5 complex but has not been structurally characterized and which might be implicated in higher order clustering (11Smith F.M. Vearing C. Lackmann M. Treutlein H. Himanen J. Chen K. Saul A. Nikolov D. Boyd A.W. J. Biol. Chem. 2004; 279: 9522-9531Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar).Although Eph receptors interact promiscuously with ephrins of the same class, they rarely interact with ephrins of the other class. A variety of factors appear to contribute to class specificity. B class Eph-ephrin interactions require considerable structural rearrangements of both the receptor and the ephrin, whereas EphA receptors and ephrin-A ligands appear to undergo smaller rearrangements when forming a complex (8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). Differences in critical residues located in the interacting and sequence in the class loop of the Eph receptors also to a in class (3Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (91) Google Scholar, 4Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (272) Google Scholar, 5Himanen 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 (368) Google Scholar, 6Chrencik J.E. Brooun A. Kraus M.L. Recht M.I. Kolatkar A.R. Han G.W. Seifert J.M. Widmer H. Auer M. Kuhn P. J. Biol. Chem. 2006; 281: 28185-28192Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7Qin H. Shi J. Noberini R. Pasquale E.B. Song J. J. Biol. Chem. 2008; 283: 29473-29484Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 8Himanen J.P. Goldgur Y. Miao H. Myshkin E. Guo H. Buck M. Nguyen M. Rajashankar K.R. Wang B. Nikolov D.B. EMBO Rep. 2009; 10: 722-728Crossref PubMed Scopus (89) Google Scholar). However, of interclass binding also can bind and EphA4 can bind all three ephrin-B ligands E.B. Nat. Neurosci. 2004; 7: PubMed Scopus Google Scholar).The structure of the ephrin-binding domain in complex with has been 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 (368) Google and that the two interact through the high affinity which is reduced with intraclass complexes, in a binding affinity. EphA4 binding to ephrin-B ligands is also to ephrin-A ligands. However, the of interclass interactions has been in biological EphA4 with formation H. P. 2005; PubMed Scopus Google Scholar), whereas EphA4 with ephrin-B2 in the and of the A. K. Biol. 7: Full Text Full Text PDF PubMed Google Scholar, Nature. PubMed Scopus Google Scholar), A. C. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and formation in the K. M. Boyd A.W. R. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. M. J. R. Dev. 2001; PubMed Scopus Google Scholar, M.I. Cowan C.A. P. P. Henkemeyer M. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, K. J.M. A. R. PubMed Scopus Google Scholar), and of by M.I. Henkemeyer M. 2005; PubMed Scopus Google Scholar).The distinctive ability of EphA4 to bind both ephrin-A and ephrin-B ligands an to the structural the promiscuity of Eph receptor-ephrin but structural has been for complexes. In we report the crystal structure of the complex and a polar region, structurally the ephrin-binding as critical for binding. also characterized the complex in solution by NMR which represents the first NMR of an Eph-ephrin complex. that EphA4 uses different strategies for binding ephrin-A ephrin-B ligands, remarkable promiscuity.