Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity
2009; Elsevier BV; Volume: 285; Issue: 1 Linguagem: Inglês
10.1074/jbc.m109.064824
ISSN1083-351X
AutoresHaina Qin, Roberta Noberini, Xuelu Huan, Jiahai Shi, Elena B. Pasquale, Jianxing Song,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoEphA 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 regions and sequence differences in the class specificity H-I loop of the Eph receptors also seem to play a role in class specificity (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, examples of interclass binding also exist: EphB2 can bind ephrin-A5, and EphA4 can bind all three ephrin-B ligands (12Pasquale E.B. Nat. Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (127) Google Scholar).The structure of the EphB2 ephrin-binding domain in complex with ephrin-A5 has been determined (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) and shows that the two proteins interact only through the high affinity interface, which is greatly reduced compared with intraclass complexes, resulting in a lower binding affinity. EphA4 binding to ephrin-B ligands is also weaker than to ephrin-A ligands. However, the physiological relevance of EphA4-ephrin-B interclass interactions has been demonstrated in many biological systems. For example, EphA4 interaction with ephrin-B1 stabilizes blood clot formation (13Prévost N. Woulfe D.S. Jiang H. Stalker T.J. Marchese P. Ruggeri Z.M. Brass L.F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9820-9825Crossref PubMed Scopus (121) Google Scholar), whereas EphA4 interaction with ephrin-B2 and/or ephrin-B3 regulates cell sorting in the rhombomeres and branchial arches of the developing hindbrain (14Smith A. Robinson V. Patel K. Wilkinson D.G. Curr. Biol. 1997; 7: 561-570Abstract Full Text Full Text PDF PubMed Google Scholar, 15Xu Q. Mellitzer G. Robinson V. Wilkinson D.G. Nature. 1999; 399: 267-271Crossref PubMed Scopus (370) Google Scholar), somite morphogenesis (16Barrios A. Poole R.J. Durbin L. Brennan C. Holder N. Wilson S.W. Curr. Biol. 2003; 13: 1571-1582Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), axon guidance and circuit formation in the developing spinal cord (17Kullander K. Mather N.K. Diella F. Dottori M. Boyd A.W. Klein R. Neuron. 2001; 29: 73-84Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 18Kullander K. Croll S.D. Zimmer M. Pan L. McClain J. Hughes V. Zabski S. DeChiara T.M. Klein R. Yancopoulos G.D. Gale N.W. Genes Dev. 2001; 15: 877-888Crossref PubMed Scopus (212) Google Scholar, 19Yokoyama N. Romero M.I. Cowan C.A. Galvan P. Helmbacher F. Charnay P. Parada L.F. Henkemeyer M. Neuron. 2001; 29: 85-97Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 20Kullander K. Butt S.J. Lebret J.M. Lundfald L. Restrepo C.E. Rydström A. Klein R. Kiehn O. Science. 2003; 299: 1889-1892Crossref PubMed Scopus (278) Google Scholar), and inhibition of axon outgrowth by myelin (21Benson M.D. Romero M.I. Lush M.E. Lu Q.R. Henkemeyer M. Parada L.F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 10694-10699Crossref PubMed Scopus (260) Google Scholar).The distinctive ability of EphA4 to bind both ephrin-A and ephrin-B ligands makes it an attractive model to understand the structural principles underlying the selectivity versus promiscuity of Eph receptor-ephrin interactions, but no structural information has been available for EphA4-ephrin complexes. In this study, we report the crystal structure of the EphA4-ephrin-B2 complex and identify a polar contact region, structurally separated from the ephrin-binding channel, as critical for EphA4-ephrin-B2 binding. We also characterized the EphA4-ephrin-B2 complex in solution by NMR spectroscopy, which represents the first NMR visualization of an Eph-ephrin complex. Interestingly, our results show that EphA4 uses different strategies for binding ephrin-A versus ephrin-B ligands, thus achieving remarkable promiscuity.DISCUSSIONThe binding promiscuity between Eph receptors and ephrin ligands appears to be a key strategy enabling Eph-ephrin signaling networks to control a wide array of biological functions. Understanding the structural principles governing promiscuity versus selectivity of Eph receptor-ephrin binding is therefore important for elucidating the mechanisms underlying the biological functions of the Eph system and is also critical for the design of antagonists to target Eph-ephrin interactions. EphA4 is the only Eph receptor that can bind with substantial affinity all ephrin-A and ephrin-B ligands (12Pasquale E.B. Nat. Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (127) Google Scholar). Here, we have reported the structure of the EphA4-ephrin-B2 complex, which represents the first structure of a complex between an EphA receptor and an ephrin-B ligand.The overall architecture of the EphA4-ephrin-B2 complex is very similar to those determined previously for other Eph-ephrin complexes. The high affinity interface of the complex can be divided into two relatively independent regions. One mostly involves the hydrophobic interactions between the EphA4 ligand-binding channel and the ephrin-B2 G-H loop. The other, which was observed in EphB-ephrin-B complexes (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) but was greatly reduced in the EphA2-ephrin-A1 complex and absent in the EphB2-ephrin-A5 complex (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), involves polar interactions between surface residues Gln12 and Glu14 in EphA4 and Gln109 and Lys112 in ephrin-B2. We did not obtain evidence for a distinct lower affinity binding interface that may mediate tetramerization of two EphA4-ephrin-B2 heterodimers similar to that described 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). Such an interface is also not evident in the EphB4-ephrin-B2, EphA2-ephrin-A1, and EphB2-ephrin-A5 complexes (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).Several notable structural variations are observed in EphA4 upon complex formation with ephrin-B2, including those in the D and E β-strands as well as the A-C, D-E, G-H, and J-K loops. Significant structural rearrangements in these regions have also been reported for EphB receptors upon 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, 42Goldgur Y. Paavilainen S. Nikolov D. Himanen J.P. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2009; 65: 71-74Crossref PubMed Scopus (7) Google Scholar). In contrast, only minor structural changes have been observed upon formation of the EphA2-ephrin-A1 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 EphA4-ephrin-B2 structure also explains the relatively weak affinity of the EphA4-ephrin-B2 interclass binding (Kd = 203 nm) compared with intraclass interactions, such as those of EphB2 with ephrin-B2 (Kd = 22 nm) and EphB4 with ephrin-B2 (Kd = 40 nm). Because of sequence variations, a hydrophobic patch present in the EphB2-ephrin-B2 complex is absent in the EphA4-ephrin-B2 complex. As a consequence, half of the EphA4 J-K loop remains open and does not make any contacts with the ephrin-B2 G-H loop (Fig. 4a). Given that the structurally equivalent EphB2 loop region is more open in the complex with ephrin-A5 and that the binding affinity of EphB2 for ephrin-A5 is even lower (Kd = 320 nm) (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), this feature likely accounts at least in part for the low binding affinity between EphA4 and ephrin-B2. However, comparing Kd values for wild-type and mutant proteins shows that the surface contacts formed between EphA4 residues Gln12 and Glu14 and ephrin-B2 residues Gln109 and Lys112 increase by ∼10-fold the binding affinity and thus play a critical role in EphA4-ephrin-B2 interclass binding.Mutation of the two EphA4 residues involved in the interface yields an EphA4 mutant that does not appear to undergo global structural changes or modifications in the ephrin-binding channel. This is evident from the unchanged binding affinity of the EphA4 mutant for two small molecule antagonists that bind within the channel. Interestingly, the polar surface contact region of EphA4 does not appear to be necessary for the intraclass binding with ephrin-A ligands, which probably involves a more intimate fit between the EphA4 ligand-binding channel and the ephrin-A G-H loop. This is the case for the EphA2-ephrin-A1 complex, where the surface contact 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). Similarly, this region is likely not present or minimal in EphA4-ephrin-A complexes.On the other hand, Gln109 and Lys112 of ephrin-B2 have been shown 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 could explain the 2–3-fold decrease in the affinity of mutant ephrin-B2 for EphB receptors. However, we cannot exclude that the decrease in binding may be due to the changes in the overall conformation of the mutant ephrin observed by CD spectroscopy. Nevertheless, the much more pronounced impairment in the binding of mutant ephrin-B2 to EphA4 than to EphB receptors suggests that the surface contact region is much more critical for the EphA4-ephrin-B2 interclass binding than the EphB-ephrin-B2 intraclass binding. Interestingly, the residue corresponding to ephrin-B2 Gln109 is a Leu in ephrin-B3 and therefore cannot be involved in the hydrogen bond with EphA4 Gln12 that is instead present in the EphA4-ephrin-B2 complex. However, the binding affinity of the EphA4 mutant for ephrin-B2 or ephrin-B3 is equally reduced compared with wild-type EphA4, suggesting that the salt bridge between ephrin-B2 Lys112 and EphA4 Gln14 might play a more important role in EphA4-ephrin-B interclass binding than the hydrogen bond between ephrin-B2 Gln109 and EphA4 Gln12.NMR characterization also provides the first insight into the dynamic aspects associated with EphA4-ephrin-B2 binding in solution, which could not be achieved from analysis of the more static crystallized complex. Interestingly, the NMR results show that many EphA4 residues perturbed upon ephrin-B2 binding (corresponding to HSQC peaks that shift or disappear) are located outside the high affinity EphA4-ephrin-B2 binding interface. For example, many residues in the EphA4 L and H strands are significantly perturbed even though they are far away from the binding interface. Interestingly, in the crystal structure, these residues do not appear to undergo any detectable conformational changes upon ephrin-B2 binding (Fig. 1b). In contrast, once the interactions mediated by EphA4 Gln12 and Glu14 are removed, the residues perturbed by the binding of ephrin-B2 are limited only to the EphA4 interface in direct contact with ephrin-B2. Thus, contacts mediated by Gln12 and/or Glu14 have far-reaching effects over the entire EphA4 ligand-binding domain. It is tempting to speculate that these perturbations, involving peak shifts and disappearances, may reflect dynamic changes in EphA4 that are initiated by ephrin-B binding and are critical for biological function. However, although we did not obtain evidence for complexes larger than heterodimers, it cannot be completely excluded that a very weak tendency might exist for EphA4-ephrin-B2 heterodimeric complexes to form high order complexes such as tetramers, in analogy to what is observed with EphB2-ephrin-B2 complexes (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). It will be interesting to investigate this possibility in future NMR studies as exemplified previously (43Jee J. Ishima R. Gronenborn A.M. J. Phys. Chem. B. 2008; 112: 6008-6012Crossref PubMed Scopus (18) Google Scholar).The new EphA4-ephrin-B2 complex structure that we have characterized, together with those determined previously for other Eph receptor-ephrin pairs, highlights a surprising diversity in the use of the two regions of the high affinity interface to accomplish intraclass or interclass Eph receptor-ephrin binding. For example, it appears that intraclass binding is mediated almost exclusively (A class) or predominantly (B class) by the hydrophobic Eph channel-ephrin G-H loop region of the interface. Accordingly, the fit of the ephrin G-H loop into the Eph channel is more intimate for the A than the B class. EphB2-ephrin-A5 interclass binding relies only on the channel region of the interface, although the loose interclass fit of the EphB channel and the ephrin-A G-H loop results in the lowest binding affinity among the complexes structurally characterized so far. Interestingly, we found that EphA4-ephrin-B2 interclass binding uses a unique strategy, where the presumably very weak binding through the Eph channel-ephrin G-H loop is supplemented by interactions in the polar surface contact region of the interface. In this manner, EphA4 achieves the highest promiscuity among the Eph receptors.As a consequence of this variability in Eph receptor-ephrin interfaces, the design of antagonists to target Eph-ephrin interactions may be more challenging than previously thought, and diverse strategies may be needed depending on the Eph receptor and the ephrin involved. Consistent with this notion, we previously found that two small molecule antagonists that target the ephrin-binding channel of EphA4 inhibit the binding of some ephrins but not others. For example, we did not detect inhibition of ephrin-B2 and ephrin-A4 binding to EphA4 at concentrations that completely inhibited the binding of other ephrins (40Noberini R. Koolpe M. Peddibhotla S. Dahl R. Su Y. Cosford N.D. Roth G.P. Pasquale E.B. J. Biol. Chem. 2008; 283: 29461-29472Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The structural information we obtained and the effects of the EphA4 and ephrin-B2 mutations also suggest that it is possible to selectively inhibit EphA4 binding to ephrin-B but not ephrin-A ligands by disrupting the polar surface region of the high affinity interface, whereas the binding of ephrin-A ligands to EphA4 may be selectively inhibited by disrupting appropriate contacts in the channel region. Such strategies may help dissect the biological roles of intraclass versus interclass EphA4-ephrin binding and guide more selective approaches for the design of EphA4 inhibitors. 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 regions and sequence differences in the class specificity H-I loop of the Eph receptors also seem to play a role in class specificity (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, examples of interclass binding also exist: EphB2 can bind ephrin-A5, and EphA4 can bind all three ephrin-B ligands (12Pasquale E.B. Nat. Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (127) Google Scholar).The structure of the EphB2 ephrin-binding domain in complex with ephrin-A5 has been determined (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) and shows that the two proteins interact only through the high affinity interface, which is greatly reduced compared with intraclass complexes, resulting in a lower binding affinity. EphA4 binding to ephrin-B ligands is also weaker than to ephrin-A ligands. However, the physiological relevance of EphA4-ephrin-B interclass interactions has been demonstrated in many biological systems. For example, EphA4 interaction with ephrin-B1 stabilizes blood clot formation (13Prévost N. Woulfe D.S. Jiang H. Stalker T.J. Marchese P. Ruggeri Z.M. Brass L.F. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9820-9825Crossref PubMed Scopus (121) Google Scholar), whereas EphA4 interaction with ephrin-B2 and/or ephrin-B3 regulates cell sorting in the rhombomeres and branchial arches of the developing hindbrain (14Smith A. Robinson V. Patel K. Wilkinson D.G. Curr. Biol. 1997; 7: 561-570Abstract Full Text Full Text PDF PubMed Google Scholar, 15Xu Q. Mellitzer G. Robinson V. Wilkinson D.G. Nature. 1999; 399: 267-271Crossref PubMed Scopus (370) Google Scholar), somite morphogenesis (16Barrios A. Poole R.J. Durbin L. Brennan C. Holder N. Wilson S.W. Curr. Biol. 2003; 13: 1571-1582Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), axon guidance and circuit formation in the developing spinal cord (17Kullander K. Mather N.K. Diella F. Dottori M. Boyd A.W. Klein R. 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U.S.A. 2005; 102: 10694-10699Crossref PubMed Scopus (260) Google Scholar).The distinctive ability of EphA4 to bind both ephrin-A and ephrin-B ligands makes it an attractive model to understand the structural principles underlying the selectivity versus promiscuity of Eph receptor-ephrin interactions, but no structural information has been available for EphA4-ephrin complexes. In this study, we report the crystal structure of the EphA4-ephrin-B2 complex and identify a polar contact region, structurally separated from the ephrin-binding channel, as critical for EphA4-ephrin-B2 binding. We also characterized the EphA4-ephrin-B2 complex in solution by NMR spectroscopy, which represents the first NMR visualization of an Eph-ephrin complex. Interestingly, our results show that EphA4 uses different strategies for binding ephrin-A versus ephrin-B ligands, thus achieving remarkable promiscuity.
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