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• W2084674165 abstract "The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of ∼15 μm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-Å resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities. The Eph family of receptor tyrosine kinases has been implicated in tumorigenesis as well as pathological forms of angiogenesis. Understanding how to modulate the interaction of Eph receptors with their ephrin ligands is therefore of critical interest for the development of therapeutics to treat cancer. Previous work identified a set of 12-mer peptides that displayed moderate binding affinity but high selectivity for the EphB2 receptor. The SNEW antagonistic peptide inhibited the interaction of EphB2 with ephrinB2, with an IC50 of ∼15 μm. To gain a better molecular understanding of how to inhibit Eph/ephrin binding, we determined the crystal structure of the EphB2 receptor in complex with the SNEW peptide to 2.3-Å resolution. The peptide binds in the hydrophobic ligand-binding cleft of the EphB2 receptor, thus competing with the ephrin ligand for receptor binding. However, the binding interactions of the SNEW peptide are markedly different from those described for the TNYL-RAW peptide, which binds to the ligand-binding cleft of EphB4, indicating a novel mode of antagonism. Nevertheless, we identified a conserved structural motif present in all known receptor/ligand interfaces, which may serve as a scaffold for the development of therapeutic leads. The EphB2-SNEW complex crystallized as a homodimer, and the residues involved in the dimerization interface are similar to those implicated in mediating tetramerization of EphB2-ephrinB2 complexes. The structure of EphB2 in complex with the SNEW peptide reveals novel binding determinants that could serve as starting points in the development of compounds that modulate Eph receptor/ephrin interactions and biological activities. The erythropoietin-producing hepatocellular carcinoma (Eph) 3The abbreviation used is: Eph, erythropoietin-producing hepatocellular carcinoma. family is the largest family of receptor tyrosine kinases identified to date, with 16 structurally similar family members (1Committee Eph Nomenclature Cell. 1997; 90: 403-404Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). The Eph family plays important roles in both developing and adult tissues, and regulates biological processes such as tissue patterning, development of the vascular system, axonal guidance, and neuronal development (2Brantley-Sieders D.M. Chen J. Angiogenesis. 2004; 7: 17-28Crossref PubMed Scopus (127) Google Scholar, 3Carmeliet P. Collen D. Curr. Top Microbiol. Immunol. 1999; 237: 133-158PubMed Google Scholar, 4Kullander K. Klein R. Nat. Rev. Mol. Cell. Biol. 2002; 3: 475-486Crossref PubMed Scopus (955) Google Scholar, 5Pasquale E.B. Nat. Rev. Mol. Cell. Biol. 2005; 6: 462-475Crossref PubMed Scopus (852) Google Scholar, 6Poliakov A. Cotrina M. Wilkinson D.G. Dev. Cell. 2004; 7: 465-480Abstract Full Text Full Text PDF PubMed Scopus (360) Google Scholar). The EphB2 receptor plays a role in the development of several tissues. The loss of EphB2 and the related EphB3 receptor, which has some redundant functions, results in cleft palate, the failure to remodel the vascular plexus, impaired heart development, defects in urorectal development, and abnormal development of dendritic spines in the hippocampus (7Adams R.H. Wilkinson G.A. Weiss C. Diella F. Gale N.W. Deutsch U. Risau W. Klein R. Genes Dev. 1999; 13: 295-306Crossref PubMed Scopus (840) Google Scholar, 8Dravis C. Yokoyama N. Chumley M.J. Cowan C.A. Silvany R.E. Shay J. Baker L.A. Henkemeyer M. Dev. Biol. 2004; 271: 272-290Crossref PubMed Scopus (193) Google Scholar, 9Henkemeyer M. Itkis O.S. Ngo M. Hickmott P.W. Ethell I.M. J. Cell Biol. 2003; 163: 1313-1326Crossref PubMed Scopus (242) Google Scholar, 10Orioli D. Henkemeyer M. Lemke G. Klein R. Pawson T. EMBO J. 1996; 15: 6035-6049Crossref PubMed Scopus (288) Google Scholar). Furthermore, some strains of EphB2 knock-out mice have defects in the inner ear (11Cowan C.A. Yokoyama N. Bianchi L.M. Henkemeyer M. Fritzsch B. Neuron. 2000; 26: 417-430Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The EphB2 receptor has also been recently found to be overexpressed in many types of cancer, including gastric (12Davalos V. Dopeso H. Velho S. Ferreira A.M. Cirnes L. Diaz-Chico N. Bilbao C. Ramirez R. Rodriguez G. Falcon O. Leon L. Niessen R.C. Keller G. Dallenbach-Hellweg G. Espin E. Armengol M. Plaja A. Perucho M. Imai K. Yamamoto H. Gebert J.F. Diaz-Chico J.C. Hofstra R.M. Woerner S.M. Seruca R. Schwartz Jr., S. Arango D. Oncogene. 2007; 26: 308-311Crossref PubMed Scopus (36) Google Scholar, 13Song J.H. Kim C.J. Cho Y.G. Kwak H.J. Nam S.W. Yoo N.J. Lee J.Y. Park W.S. Apmis. 2007; 115: 164-168Crossref PubMed Scopus (6) Google Scholar), colorectal (14Alazzouzi H. Davalos V. Kokko A. Domingo E. Woerner S.M. Wilson A.J. Konrad L. Laiho P. Espin E. Armengol M. Imai K. Yamamoto H. Mariadason J.M. Gebert J.F. Aaltonen L.A. Schwartz Jr., S. Arango D. Cancer Res. 2005; 65: 10170-10173Crossref PubMed Scopus (78) Google Scholar, 15Guo D.L. Zhang J. Yuen S.T. Tsui W.Y. Chan A.S. Ho C. Ji J. Leung S.Y. Chen X. Carcinogenesis. 2006; 27: 454-464Crossref PubMed Scopus (98) Google Scholar, 16Jubb A.M. Zhong F. Bheddah S. Grabsch H.I. Frantz G.D. Mueller W. Kavi V. Quirke P. Polakis P. Koeppen H. Clin. Cancer Res. 2005; 11: 5181-5187Crossref PubMed Scopus (92) Google Scholar, 17Batlle E. Henderson J.T. Beghtel H. van den Born M.M. Sancho E. Huls G. Meeldijk J. Robertson J. van de Wetering M. Pawson T. Clevers H. Cell. 2002; 111: 251-263Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 18Batlle E. Bacani J. Begthel H. Jonkheer S. Gregorieff A. van de Born M. Malats N. Sancho E. Boon E. Pawson T. Gallinger S. Pals S. Clevers H. Nature. 2005; 435: 1126-1130Crossref PubMed Scopus (343) Google Scholar), ovarian (19Wu Q. Suo Z. Kristensen G.B. Baekelandt M. Nesland J.M. Gynecol. Oncol. 2006; 102: 15-21Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), breast (20Wu Q. Suo Z. Risberg B. Karlsson M.G. Villman K. Nesland J.M. Pathol. Oncol. Res. 2004; 10: 26-33Crossref PubMed Scopus (101) Google Scholar), and prostate cancers (21Dong J.T. J. Cell. Biochem. 2006; 97: 433-447Crossref PubMed Scopus (174) Google Scholar, 22Kittles R.A. Baffoe-Bonnie A.B. Moses T.Y. Robbins C.M. Ahaghotu C. Huusko P. Pettaway C. Vijayakumar S. Bennett J. Hoke G. Mason T. Weinrich S. Trent J.M. Collins F.S. Mousses S. Bailey-Wilson J. Furbert-Harris P. Dunston G. Powell I.J. Carpten J.D. J. Med. Genet. 2006; 43: 507-511Crossref PubMed Scopus (54) Google Scholar), and glioblastoma (23Nakada M. Niska J.A. Tran N.L. McDonough W.S. Berens M.E. Am. J. Pathol. 2005; 167: 565-576Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 24Nakada M. Niska J.A. Miyamori H. McDonough W.S. Wu J. Sato H. Berens M.E. Cancer Res. 2004; 64: 3179-3185Crossref PubMed Scopus (148) Google Scholar). In some tumor types EphB2 appears to have tumor promoting effects, whereas in others it has tumor suppressor effects (17Batlle E. Henderson J.T. Beghtel H. van den Born M.M. Sancho E. Huls G. Meeldijk J. Robertson J. van de Wetering M. Pawson T. Clevers H. Cell. 2002; 111: 251-263Abstract Full Text Full Text PDF PubMed Scopus (941) Google Scholar, 18Batlle E. Bacani J. Begthel H. Jonkheer S. Gregorieff A. van de Born M. Malats N. Sancho E. Boon E. Pawson T. Gallinger S. Pals S. Clevers H. Nature. 2005; 435: 1126-1130Crossref PubMed Scopus (343) Google Scholar). In addition, similar to EphB4, EphB2 on the surface of tumor cells may promote tumor angiogenesis by interacting with ephrinB2 in tumor blood vessels (25Noren N.K. Lu M. Freeman A.L. Koolpe M. Pasquale E.B. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5583-5588Crossref PubMed Scopus (218) Google Scholar). Therefore, targeting EphB2 represents a promising avenue for therapeutic intervention. Peptides and chemical compounds that bind to EphB2 may be used to target anti-cancer agents to EphB2-expressing tumors (26Mao W. Luis E. Ross S. Silva J. Tan C. Crowley C. Chui C. Franz G. Senter P. Koeppen H. Polakis P. Cancer Res. 2004; 64: 781-788Crossref PubMed Scopus (113) Google Scholar) and, in certain tumor types, interfering with EphB2 ligand binding should inhibit tumor progression and tumor angiogenesis. The Eph receptors and the ephrin ligands are classified into two major classes, A or B, based on their sequence identities and ligand binding preferences. Class A receptors (EphA1-EphA10) bind preferentially to ephrinA ligands (ephrinA1-ephrinA6), whereas class B receptors (EphB1–EphB6) bind preferentially to ephrinB ligands (ephrinB1–ephrinB3) (27Gale 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 (758) Google Scholar). Although the Eph receptors bind promiscuously the ephrins of the same A or B class, interactions across classes are rare (28Pasquale E.B. Nat. Neurosci. 2004; 7: 417-418Crossref PubMed Scopus (128) Google Scholar). An interesting feature of the Eph/ephrin interaction is the fact that both the receptor and the ligand are membrane bound, and are capable of transducing signals bidirectionally either in the forward direction through the Eph receptor cytoplasmic domain, or in the reverse direction through the ephrin, to elicit a series of biological responses in both receptor- and ephrin-expressing cells. The domain architecture of the Eph receptors is highly conserved. The cytoplasmic region of the receptors consists of a juxtamembrane region, a kinase domain, a SAM domain (sterile α-domain), and a PDZ binding motif (PSD-95 post-synaptic density protein, Discs large, and Zona occludens tight junction protein). Extracellularly, the receptors contain a ligand-binding domain at the N terminus, a cysteine-rich region, and two fibronectin type III repeats. The ligand-binding domain of the Eph receptor has been characterized as the minimal region required for high affinity interaction with the ephrins (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar). The ephrin ligands also have a structurally conserved extracellular domain characterized by a Greek-key topology. The ligands deviate, however, in their attachment to the cell membrane. The ephrinB ligands contain a transmembrane region, whereas the ephrinA ligands are anchored to the membrane by a glycosylphosphatidylinositol linkage. Previously reported crystal structures of Eph receptors in complex with a ligand revealed a heterodimerization interface on the surface of the Eph receptor ligand-binding domain that mediates a high affinity receptor/ligand interaction. In addition, a lower affinity tetramerization interface was identified in EphB2-ephrinB2 complex crystals that mediated the multimerization of EphB2 receptor/ephrinB2 dimers (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar). In the high affinity interface, the G–H loop of the ephrin inserts into the hydrophobic ligand-binding cavity formed by the D–E, G–H, and J–K loops of the Eph receptor, resulting in a nanomolar binding affinity (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar, 30Chrencik 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 (78) Google Scholar). Previously, the crystal structure of the EphB4 receptor in complex with the high affinity (40 nm) and antagonistic TNYL-RAW (TNYLFSPNGPIARAW) peptide was elucidated (31Chrencik 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 (77) Google Scholar). The structure revealed that the antagonistic peptide binds within the EphB4 ligand binding cavity, precluding interaction with the G–H loop of ephrinB2. Phage display screens have identified a series of 12-mer peptides that target the ligand-binding domains of several Eph receptors and antagonize their interactions with ephrins, whereas maintaining exceptional specificity for a particular Eph receptor (32Koolpe M. Burgess R. Dail M. Pasquale E.B. J. Biol. Chem. 2005; 280: 17301-17311Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). One peptide, SNEW (SNEWIQPRLPQH), displayed moderate affinity and high selectivity for the EphB2 receptor, and inhibited EphB2 signaling pathways in cells (32Koolpe M. Burgess R. Dail M. Pasquale E.B. J. Biol. Chem. 2005; 280: 17301-17311Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 33Ogawa K. Wada H. Okada N. Harada I. Nakajima T. Pasquale E.B. Tsuyama S. J. Cell Sci. 2006; 119: 559-570Crossref PubMed Scopus (42) Google Scholar, 34Dail M. Richter M. Godement P. Pasquale E.B. J. Cell Sci. 2006; 119: 1244-1254Crossref PubMed Scopus (61) Google Scholar) and the formation of capillary tube-like structures by human umbilical vein endothelial cells (35Salvucci O. de la Luz Sierra M. Martina J.A. McCormick P.J. Tosato G. Blood. 2006; 108: 2914-2922Crossref PubMed Scopus (78) Google Scholar). To understand the molecular determinants of the EphB2-SNEW interaction, we determined the crystal structure of the complex at 2.3-Å resolution. The structure reveals that the peptide binds as expected in the hydrophobic ligand binding cleft of the receptor, explaining its ability to inhibit ephrin binding. The overall binding interface of the SNEW peptide is small, and dominated by hydrophobic interactions and weak polar interactions. Thermodynamic characterization of the EphB2 ligand-binding domain-SNEW complex revealed a low micromolar binding affinity, consistent with the weak interaction network observed in the crystal structure. Comparison of the EphB2-SNEW complex with the EphB4/TNYL-RAW complex revealed significant differences in the binding interfaces of the two peptides. However, a conserved structural motif (Ile or Pro) has now been identified in all known Eph receptor/ligand crystal structures. This motif has a critical role in stabilizing a conserved disulfide bridge in the Eph receptor. Together with previously available structural information, our data should accelerate drug discovery efforts aimed at targeting the extracellular Eph/ephrin protein-protein interaction. Protein Expression and Purification—The human EphB2 construct was designed based on similarity with the human EphB4 receptor previously described. EphB2-(28–203) was expressed in baculovirus in Hi5 insect cells as described elsewhere (30Chrencik 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 (78) Google Scholar, 31Chrencik 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 (77) Google Scholar). Media containing secreted EphB2 was concentrated and buffer exchanged using a Hydrosart Crossflow filter (Sartorius, NY). The secreted protein was bound to Probond IMAC resin (Invitrogen), washed with 50 mm Tris, pH 7.8, 400 mm NaCl, 20 mm imidazole buffer and eluted with 50 mm Tris, pH 7.8, 400 mm NaCl, 250 mm imidazole. Following IMAC purification, the receptor was concentrated to 5 mg/ml and loaded onto a Phenomenex S2000 column (Phenomenex, CA) to remove aggregated material. Crystallization and Data Collection—The EphB2 receptor was concentrated to 5 mg/ml in 25 mm Hepes, pH 7.2, 150 mm NaCl, and 1 mm CaCl2 in the presence of a 5-fold molar excess of SNEW peptide (SNEWIQPRLPQH, Biopeptide, Inc.), and crystallized by sitting drop vapor diffusion at 20 °C against a reservoir of 100 mm Hepes, pH 7.2, 100 mm ammonium sulfate, and 20% PEG-3350. The crystals were cryoprotected in 25% propylene glycol, and flash-frozen in liquid nitrogen. Crystals of the EphB2-SNEW complex formed in the P32 space group (a = b = 40.18, c = 238.03). A single crystal diffracted to 2.3-Å resolution at 100 K on the GM/CA-CAT beamline at the Advanced Photon Source (Argonne). Data were processed and reduced using the HKL2000 package (36Otwinowski Z. Minor W. Carter Jr., C. Sweet R. Macromolecular Crystallography, Part A. Academic Press, New York1997: 307-326Google Scholar). The calculated Matthews coefficient (VM) suggested that the EphB2-SNEW complex existed as a dimer. The structure was determined by molecular replacement with MolRep (CCP4i) (37P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) using the structure of apo-EphB2 (Protein Data Bank code 1NUK (38Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (92) Google Scholar)) as a search model. The structure was refined with Refmac5 (CCP4i) using torsion angle dynamics and the maximum likelihood function target (Table 1), and manual model building performed in Coot (37P4 CC Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar, 39Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar, 40Vagin A.A. Steiner R.A. Lebedev A.A. Potterton L. McNicholas S. Long F. Murshudov G.N. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2184-2195Crossref PubMed Scopus (1078) Google Scholar). All 176 residues of EphB2, as well as 11 of 12 residues from the SNEW peptide, were readily modeled into the electron density. The final structure exhibits good geometry with no Ramachandran outliers as per Molprobity (41Lovell S.C. Davis I.W. Arendall 3rd, W.B. de Bakker P.I. Word J.M. Prisant M.G. Richardson J.S. Richardson D.C. Proteins. 2003; 50: 437-450Crossref PubMed Scopus (3892) Google Scholar). A crystallographic analysis with refinement statistics are described in Table 1.TABLE 1Crystallographic statistics for the EphB2-SNEW complexDataResolution (Å)aNumber in parentheses is for the highest shell35-2.3 (2.4-2.3)Space groupP32Unit cell dimensions (Å)a = b = 40.2; c = 235α = β = 90; γ = 120Completeness (%)97.0 (87.1)Rsym (%)bRsym = Σ|I〈I〉|/ΣI, where I is the observed intensity and 〈I〉 is the average intensity of multiple symmetry-related observations of that reflection7.1 (16.7)I/σ38.1 (10.5)Mean redundancy6.2 (6.6)No. reflections (unique)24,560Rcryst (%)cRcryst = Σ||Fobs| - |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors. Rsym = Σ|I - 〈I〉Σ|/I, where I is the observed intensity and 〈I〉 is the average intensity of multiple symmetry-related observations of that reflection19.8 (23.5)Rfree (%)dRfree = Σ||Fobs| - |Fcalc||/Σ|Fobs| for 10% of the data not used at any stage of structural refinement26.9 (31.3)Root mean square deviations Bond length (Å)0.02 Bond angle (°)2.1Average B factor (Å2)33.6Number of atoms Protein2812 Solvent175 Peptide174 Sulfate12a Number in parentheses is for the highest shellb Rsym = Σ|I〈I〉|/ΣI, where I is the observed intensity and 〈I〉 is the average intensity of multiple symmetry-related observations of that reflectionc Rcryst = Σ||Fobs| - |Fcalc||/Σ|Fobs|, where Fobs and Fcalc are the observed and calculated structure factors. Rsym = Σ|I - 〈I〉Σ|/I, where I is the observed intensity and 〈I〉 is the average intensity of multiple symmetry-related observations of that reflectiond Rfree = Σ||Fobs| - |Fcalc||/Σ|Fobs| for 10% of the data not used at any stage of structural refinement Open table in a new tab Isothermal Titration Calorimetry—EphB2 was dialyzed into 50 mm Hepes, pH 7.2 (at 25 °C), 150 mm NaCl, 1 mm CaCl2, prior to use in calorimetry experiments. The SNEW peptide and all mutant peptides were dissolved in the dialysis buffer. All ITC experiments were performed with a Microcal MCS ITC at 25 °C. ITC experiments were performed as described previously (30Chrencik 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 (78) Google Scholar, 31Chrencik 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 (77) Google Scholar). In Silico Combinatorial Mutagenesis—All in silico mutagenesis analyses were conducted with the ICM program (42Abagyan R. Totrov M. J. Mol. Biol. 1994; 235: 983-1002Crossref PubMed Scopus (820) Google Scholar) and incorporated the SNEW structure as a template. First, the structure was regularized with a multistep procedure. This consists of creating full-atom geometrical approximation model, rotational positioning of methyl groups, iterative optimization of geometry, and energy of the whole structure, and adjustment of polar hydrogen positions. Finally, free minimization was performed to relieve any bad contacts and check the consistency of the resulting structure. Next, residues 1 through 9 of the peptide were each mutated to all 20 amino acids individually, resulting in 120 new peptides. After each mutation, a full energy optimization of the new side chain was then performed using the Monte Carlo procedure. The associated energy includes van der Waals terms, a hydrophobicity term based on the solvent accessible surface buried upon binding, a solvation electrostatic term using a boundary-element solution of the Poisson equation, a hydrogen-bond interaction term, and the entropic contribution (43Totrov M. Abagyan R. Biopolymers. 2001; 60: 124-133Crossref PubMed Scopus (116) Google Scholar). For the double and triple mutants, three peptide mutation positions, 3, 6, and 8, were kept constant based on the highest scoring candidates from the previous round. The combinatorial approach was then repeated with the remaining positions in a similar manner as the first round. Finally, an empirical scoring function between the receptor and each peptide was calculated and stored in a table. Six single mutants, two double mutants, and a triple mutant peptide were then selected based on high scores and rational feasibility, to be synthesized and tested by ITC. The overall structure of the human EphB2 ligand-binding domain in complex with the antagonistic SNEW peptide was refined to a 2.3-Å resolution and a free R factor of 27%. The structure of EphB2 in complex with SNEW is similar to that of the apo-EphB2 receptor (38Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (92) Google Scholar), and consists of a jellyroll folding topology with 13 β-strands arranged into 2 antiparallel β-sheets forming a compact β-sandwich (Fig. 1). The β-sheets are connected by loops that vary in amino acid number and are characterized by a high degree of flexibility. For example, the D–E and J–K loops are disordered in the apo-EphB2 structure due to their inherent flexibility. However, binding of the antagonistic SNEW peptide promotes the ordering of these loops. The EphB2 structure is further stabilized by two conserved disulfide bridges, one in the G–H loop, and the other in the E–F/L–M loops (Cys105/Cys115 and Cys70/Cys192, respectively). The peptide-bound and apo-EphB2 structures superpose well, with an overall root mean square deviation of 1.7 Å over 178 respective Cα atoms. Furthermore, the EphB2-SNEW complex superposes well with respect to the ephrinB2- and ephrinA5-bound EphB2 structures, with root mean square deviations of 2.7 and 1.9 Å, respectively. Although the core of the EphB2 ligand-binding domain remains unchanged in the apo and peptide-bound structures, as well as the peptide-bound EphB4 structure, there are notable differences in the surface-exposed loop regions, particularly the D–E, G–H, and J–K loops (Fig. 2) (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar, 30Chrencik 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 (78) Google Scholar, 31Chrencik 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 (77) Google Scholar, 38Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (92) Google Scholar). Interestingly, the J–K loops from the EphB2-bound structures (PDB code 1NUK and 1KGY) are generally positioned toward the D–E loop, whereas the J–K loops from the EphB4-bound structures (PDB codes 2BBA and 2HLE) are positioned toward the G–H loop (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar, 30Chrencik 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 (78) Google Scholar, 31Chrencik 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 (77) Google Scholar, 38Himanen J.P. Henkemeyer M. Nikolov D.B. Nature. 1998; 396: 486-491Crossref PubMed Scopus (92) Google Scholar)}. The J–K loop from the EphB2-SNEW complex is positioned between these two formations, which is a result of the N-terminal residues of the SNEW peptide, which sterically precludes the J–K loop from occupying the area closer the D–E loop, as in the EphB2/ephrinB2 structure (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar). The structural flexibility of these loops in several ligand-bound EphB forms has been well documented, and it allows the receptors to use an induced fit mechanism to recognize and accommodate cognate ligands (29Himanen J.P. Rajashankar K.R. Lackmann M. Cowan C.A. Henkemeyer M. Nikolov D.B. Nature. 2001; 414: 933-938Crossref PubMed Scopus (275) Google Scholar, 30Chrencik 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 (78) Google Scholar, 44Himanen 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 (372) Google Scholar). SNEW Binding—The SNEW peptide was readily placed into the electron density after one round of refinement using |Fobs| - |Fcalc|, Φcalc maps (Fig. 3). Similar to the antagonistic TNYL-RAW peptide that binds to the EphB4 receptor, the antagonistic SNEW peptide resides in the ephrin binding cavity otherwise occupied by the long and hydrophobic G–H loop of the ephrin ligand. The SNEW peptide adopts an extended structure with no regular secondary structure elements. The N-terminal end of the peptide binds at the top of the receptor binding cleft, between the J–K and G–H loops. The position of the peptide at the junction of the J–K loop likely results in its ordering. The 12-mer peptide binds across the β-sheet floor formed by strands D and E of the receptor, forming a small network of interactions with residues lining the receptor binding cavity. The C-terminal end of the peptide emerges at the tip of the C–D and E–F loops, forming side chain/main chain interactions with residues in these loop regions. The last amino acid of the peptide, His12, could not be readily modeled into the electron" @default.
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