The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. In the present study we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrated that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical-shift changes observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4, binding. The peptides exhibit a long half-life in cell culture medium which, with their substantial binding affinity and selectivity for EphA4, makes them excellent research tools to modulate EphA4 function.
- nerve regeneration
- receptor tyrosine kinase
The Eph receptors are a large family of receptor tyrosine kinases with many functions in physiology and disease . They bind their activating ligands, the ephrins, mainly through a high-affinity binding pocket located in the N-terminal ephrin-binding domain [2,3]. A cysteine-rich region and two fibronectin type III domains connect the ephrin-binding domain to the transmembrane segment. The cytoplasmic portion of the Eph receptors includes a juxtamembrane segment, the kinase domain, a SAM (sterile-α-motif) domain and a C-terminal PDZ domain-binding motif. Interaction between Eph receptors and ephrin ligands, which are attached to the cell surface through a GPI (glycosylphosphatidylinositol)-anchor (ephrin-As) or a transmembrane domain (ephrin-Bs), typically occurs at sites of cell–cell contact. Ephrin binding promotes activation of the receptor's kinase domain, triggering ‘forward’ signals . Ephrin ligands engaged with Eph receptors can also affect the cells in which they are expressed by mediating ‘reverse’ signals.
EphA4 is highly expressed in the nervous system. The repulsive effects of EphA4 in neurons help to guide the growth of developing axons towards their synaptic targets and may contribute to inhibition of axon regeneration following injury [5–12]. In addition, EphA4 is highly expressed in adult hippocampal neurons, where it controls synaptic morphology and plasticity [13–18], and experiments in mice suggest a role for EphA4 in the behavioural responses to cocaine administration . Other evidence suggests that EphA4 contributes to maintain brain neural stem cells in an undifferentiated state . This is in contrast with muscle, where EphA4 may contribute to myoblast differentiation . Finally, increasing evidence suggests a possible role for EphA4 in several types of cancer, including glioblastoma, gastric, pancreatic, prostate and breast cancer [22–28]. EphA4 is also highly up-regulated in Sézary syndrome, a leukaemic variant of cutaneous T-cell lymphomas . Hence inhibiting the EphA4–ephrin interaction could be useful for promoting axon regeneration and regulating synaptic plasticity in the nervous system, as well as inhibiting the progression of some types of cancer.
Short peptides and small molecules that antagonize the Eph receptor–ephrin interaction represent useful tools to interfere with the Eph receptor/ephrin system [30,31]. An advantage of these artificial ligands is that they can be much more selective than the physiological ephrin ligands. Each of the five ephrin-A ligands can bind to most of the nine EphA receptors and each of the three ephrin-B ligands can bind to the five EphB receptors, whereas peptides that target only a single Eph receptor have been identified [13,32,33]. The EphA4 receptor is particularly promiscuous and can bind both ephrin-A and ephrin-B ligands [34–37], as well as a number of peptides and small molecules identified in various screens [13,38–40]. Consistent with its ability to bind diverse ligands, the ephrin-binding pocket of EphA4 can assume multiple conformations [41,42].
By screening an M13 phage display library using the entire extracellular domain of mouse EphA4 fused to the Fc portion of human IgG1 and a C-terminal histidine tag, we previously identified four 12 amino-acid-long peptides that when displayed on phage bind selectively to EphA4 and not other Eph receptors . Three of the corresponding synthetic peptides, including KYL (KYLPYWPVLSSL), APY (APYCVYRGSWSC) and VTM (VTMEAINLAFPG), also inhibit the EphA4–ephrin-A5 interaction in ELISA assays. The KYL peptide, which appeared to bind best to EphA4, has since been used in organotypic cultures to implicate EphA4 in chicken neural crest cell migration and mouse hippocampal axon arborization, as well as in adhesion assays to demonstrate the importance of the receptor in integrin-dependent adhesion of human T-cells [13,43,44]. KYL was also shown to prevent growth cone collapse in chicken retinal explants and dissociated cultures of rat cortical neurons [6,38], promote nerve regeneration and functional recovery in a rat model of spinal cord injury , and inhibit the adhesion of human T-cells to endothelial cells . Thus KYL can target human, mouse, rat and chicken EphA4 and may be useful for promoting nerve regeneration after injury and modulating immune responses.
The molecular features of the interaction of KYL, APY and VTM with EphA4 have not been previously elucidated and it was not known whether the three peptides share the same EphA4-binding interface or interact with the receptor in distinctive manners. In the present paper we report that all three peptides target the ephrin-binding pocket of EphA4 and appear to interact with partially overlapping, but distinct, interfaces. Interestingly, several EphA4 residues that are essential for ephrin-A5 ligand binding are not critical for interaction with the peptides, suggesting differences in the mode of binding of the peptide ligands and the ephrins.
KYL, APY and VTM (>95% pure) were purchased from GeneScript. The peptides used for alanine scanning were also purchased from GeneScript with a minimum purity of 85%. The identity of the peptides and peptide purity were verified by HPLC and MS. Biotinylated peptides containing a C-terminal GSGSK linker with biotin attached to the lysine side chain were synthesized using Fmoc (fluoren-9-ylmethoxycarbonyl) chemistry and purified by HPLC as described previously . Peptides were dissolved in DMSO at a concentration of 10 mM and for the KYL and APY peptides the concentration was verified by measuring the absorbance at 280 nm. The KYL peptide used for NMR analysis was produced in bacteria. A synthetic DNA sequence encoding the peptide was ligated into the plasmid pGEX-4T1 (GE Healthcare) and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) cells. The bacteria were grown at 37°C until D600 reached 0.6, induced overnight with 0.3 mM IPTG (isopropyl β-D-thiogalactopyranoside) and then harvested and lysed by sonication. The GST (glutathione transferase)-fused KYL peptide was purified by affinity chromatography with glutathione–Sepharose beads (GE Healthcare), released by on-gel cleavage with thrombin (yielding GS-KYL, KYL with N-terminal glycine and serine residues), followed by further purification by HPLC on an RP-18 column (Vydac).
Isothermal titration calorimetry
The EphA4 ephrin-binding domain (residues 29–209) was produced as described previously . Briefly, a modified pET32a vector construct encoding residues 29–209 of EphA4 (GenBank® accession number NP_004429) was used for protein expression in E. coli Rosetta-gami B (EMD Biosciences) cells (Novagen). EphA4 was purified by affinity chromatography using Ni-NTA (Ni2+-nitrilacetate) resin (Qiagen) followed by thrombin cleavage and subsequent size-exclusion chromatography (Superdex 200, GE Healthcare) in 20 mM Tris/HCL (pH 8.0) and 100 mM NaCl. The buffer was then exchanged to 10 mM Hepes (pH 7.6) and 100 mM NaCl using a PD10 desalting column (GE Healthcare). Both the EphA4 ephrin-binding domain and the peptides were diluted to obtain a final buffer containing 5% DMSO in 10 mM Hepes (pH 7.6) and 100 mM NaCl. Isothermal titration calorimetry experiments were carried out using an ITC200 calorimeter (Microcal). Aliquots (2 μl) of a solution containing one of the peptides KYL, VTM or APY at a concentration of 1 mM were injected into the cell containing 205 μl of EphA4 ephrin-binding domain solution at a concentration of 65–95 μM. Experimental data were analysed using the Origin software package from Microcal.
The construct encoding the ephrin-binding domain of human EphA4 fused to AP (alkaline phosphatase)  was mutated using the QuikChange® site-directed mutagenesis kit (Stratagene) following the manufacturer's instructions. HEK (human embryonic kidney)-293T cells were grown in DMEM (Dulbecco's modified Eagle's medium) with 10% FBS (fetal bovine serum), 1 mM sodium pyruvate and penicillin/streptomycin. Wild-type and mutant EphA4 AP proteins were produced after transfection of the cells with Lipofectamine™ 2000 (Invitrogen). Transfected cells were passaged and the medium was changed to Opti-MEM (Gibco) when the cells reached ~70% confluence. Culture medium containing the secreted EphA4 AP proteins was collected after 1 day and then again 1 day later and concentrated approximately 50-fold using Amicon Ultra-15 Centrifugal filters (Millipore). The protein concentration was estimated based on AP activity [46,47].
To measure the binding of wild-type and mutant EphA4 AP to ephrin-A5 Fc, ephrin-A5 Fc (R&D Systems) was immobilized at 0.5 μg/ml on Protein A-coated 96-well plates (Thermo Scientific) for 1 h at room temperature (22 °C) in TBS [Tris-buffered saline; 50 mM Tris/HCl and 150 mM NaCl (pH 7.5)] with 0.01% Tween 20. The plates were washed three times in TBS with 0.01% Tween 20 and EphA4 AP was added for 1 h. After washing away unbound EphA4 AP, the amount of bound EphA4 AP was quantified by using p-nitrophenyl phosphate (Thermo Scientific) diluted in SEAP buffer [105 mM diethanolamine and 0.5 mM MgCl2 (pH 9.8)] as the substrate and measuring absorbance at 405 nm. Absorbance from wells coated with Fc alone was subtracted as background.
To measure inhibition of EphA4–ephrin-A5 binding by KYL, APY or VTM peptides, different concentrations of the peptides were incubated for 3 h together with 0.05 nM wild-type or mutant EphA4 AP in Protein A-coated wells on which 1 μg/ml ephrin-A5 Fc had been previously immobilized. The only exception were the ELISAs to test the VTM peptides with alanine/serine residue replacements, which were carried out using 1 μg/ml EphA4 Fc immobilized on Protein A-coated 96-well plates and ephrin-A5 AP  at 0.05 nM.
To measure the binding of wild-type or mutant EphA4 AP to the biotinylated peptides, polystyrene high-capacity binding plates (Corning Life Sciences) where coated with 2 μg/ml streptavidin (Thermo Scientific) diluted in borate buffer [100 mM boric acid and 100 mM sodium borate (pH 8.7)] for 18 h at room temperature. The unbound streptavidin was washed away with binding buffer [50 mM Tris/HCl and 150 mM NaCl (pH 7.5) with 0.01% Tween 20 and 1 mM CaCl2] and the wells were blocked with 0.5% BSA for 1 h at room temperature. The plates were then washed three times with binding buffer and 0.5 μM biotinylated KYL, 2 μM biotinylated APY or 4 μM biotinylated VTM peptide in binding buffer were immobilized on the plates by overnight incubation at 4°C. The coated wells were then washed with binding buffer before the addition of EphA4 AP fusion proteins for 1 h at room temperature. After washing away unbound EphA4 AP, 1 mg/ml p-nitrophenil phosphate substrate in SEAP buffer was added and the absorbance at 405 nm was measured. Absorbance from the wells without peptide was subtracted as background.
NMR characterization of the EphA4–KYL complex
To characterize the binding of the KYL peptide to the ephrin-binding domain of EphA4 by NMR spectroscopy, two-dimensional 1H-15N HSQC (heteronuclear single-quantum coherence) spectra of the 15N-labelled EphA4 ephrin-binding domain were acquired at an EphA4 concentration of 100 μM in the absence or presence of the KYL peptide at several molar ratios (1:0.5, 1:1, 1:1.5, 1:2, 1:2.5 and 1:3 EphA4/KYL). Consistent with the high KYL-binding affinity measured by isothermal titration calorimetry, we found that at an EphA4/KYL ratio of 1:1 most EphA4 HSQC peaks were already converted into those corresponding to the receptor in complex with KYL, and further increases in KYL concentration did not cause additional changes. Therefore we could not assign the HSQC peaks for the EphA4–KYL complex by following progressive peak shifts as we did in the previous characterization of the interaction of the EphA4 ephrin-binding domain with small molecules . Instead, we achieved the sequential assignments for the EphA4 ephrin-binding domain in complex with KYL by analysing triple-resonance HNCACB and CBCACONH spectra acquired using a 15N-13C double-labelled sample in the presence of the unlabelled KYL peptide at an EphA4/KYL ratio of 1:1.5. The degree of perturbation was measured by an integrated chemical-shift index calculated from the formula [(ΔH)2+(ΔN)2/5]1/2.
Molecular docking for the KYL peptide and the EphA4 ephrin-binding domain was performed by using the software HADDOCK (high-ambiguity-driven protein–protein docking) v1.3, which can use NMR chemical-shift perturbation data and mutagenesis data to derive the docking while allowing various degrees of flexibility. The docking procedure was performed in three steps. First, randomization and rigid body energy minimization; secondly, semi-flexible simulated annealing; and thirdly, flexible explicit solvent refinement.
According to the HADDOCK definition, the solution-accessible residues of the EphA4 ephrin-binding domain with larger chemical-shift perturbation values (>0.3 p.p.m.) were set as active residues. Two additional EphA4 residues, Val129 and Arg134, were also set as active residues on the basis of the results of the mutagenesis experiments. EphA4 Val129 could not be assigned by NMR even in the unbound EphA4 and the HSQC peak for Arg134 disappears upon formation of the EphA4–KYL complex. Furthermore, KYL residues Lys1, Tyr2, Trp6–Leu9 and Leu12 were also set as active residues on the basis of the results of the alanine scan.
The PDB file (code 3CKH) for GS-KYL was generated by the program CNS from the sequence of the peptide: 1000 structures were generated during the rigid-body docking, and the best 100 structures were selected for semi-flexible simulated annealing. The best 20 structures among those were selected for further refinement according to the explicit water model, which treats water as individual solvent molecules. Docking solutions were ranked based on the average HADDOCK score. The best model of the complex was selected for further analysis and displayed by PyMOL (http://www.pymol.org).
Pull-down assays with KYL
Mouse brain or a previously described B35 neuroblastoma cell clone stably transfected with chicken EphA4  were homogenized in Hepes lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM KCl and 1 mM EDTA] containing phosphatase and protease inhibitors. Then 3–10 μg of biotinylated peptides or a biotinylated control peptide immobilized on streptavidin beads were incubated overnight at 4°C with 4 mg of brain lysate or cell lysate derived from 1/5 of a nearly confluent 10-cm plate, washed several times with Hepes lysis buffer and boiled in sample buffer. Eluted proteins were separated by SDS/PAGE (4–20% gradient gel) and probed by immunoblotting for EphA4.
Cell imaging with KYL bound to fluorescent quantum dots
COS cells were grown in DMEM with 10% FBS, 1 mM sodium pyruvate and penicillin/streptomycin and transfected using SuperFect Transfection Reagent (Qiagen) with a pEGFP plasmid (Clontech) encoding the chicken EphA4 extracellular and transmembrane portions (amino acids 1–576) fused to EGFP (enhanced green fluorescent protein), which replaces the cytoplasmic domain, or with the pEGFP-F vector (Clontech), which encodes the membrane-targeted farnesylated EGFP. At 1 day after transfection, the cells were plated on to glass coverslips precoated with 0.1 mg/ml fibronectin and labelled 1 day later. To label the cells, 500 nM biotinylated KYL and 20 nM streptavidin 655 nm quantum dots (Qdots, Invitrogen) were incubated together in binding buffer (PBS with 1 mM CaCl2 and 2% FBS) for 20 min on ice. The cells were then incubated on ice with the KYL peptide bound to quantum dots for 20 min, washed with ice-cold PBS with 1 mM CaCl2, and fixed in 4% paraformaldehyde and 4% sucrose in PBS for 10 min at room temperature. The cells were then washed again with ice-cold PBS with 1 mM CaCl2, and permeabilized in PBS containing 0.05% Triton X-100 for 5 min. Permeabilized cells were washed with ice-cold PBS and stained with DAPI (4′,6-diamidino-2-phenylindole) for 10 min at room temperature. Coverslips were mounted on to glass slides using Pro-long Gold (Molecular Probes) and imaged under a fluorescence microscope.
Determination of peptide stability
Peptides were incubated at 37°C in medium conditioned for 3 days by subconfluent to confluent PC3 prostate cancer cells or C2C12 myoblasts, or in mouse serum at concentrations of 100 μM for KYL, 200 μM for APY or 500 μM for VTM. Aliquots were collected at different time points and used in ELISA measuring inhibition of EphA4 AP–ephrin-A5 Fc binding. For these assays, ephrin-A5 Fc was immobilized at 1 μg/ml for 1 h at room temperature in Protein A-coated 96-well plates as described above. Conditioned medium or serum containing the peptides were incubated in the wells at a 1:20 dilution (corresponding to final concentrations of 5 μM for KYL, 10 μM for APY and 25 μM for VTM in the absence of proteolytic degradation) with 0.05 nM EphA4 AP for 30 min at 4°C. These peptide concentrations yield ~80% inhibition of EphA4 AP binding to ephrin-A5 Fc. The amount of bound AP fusion protein was quantified as described above. The absorbance obtained from wells coated with Fc and incubated with EphA4 AP and medium or serum was subtracted as the background. The absorbance obtained from wells incubated with conditioned medium or mouse serum not containing any peptide was used to determine the 0% inhibition level (efficacy=0) and the absorbance in the presence of peptide not incubated in medium or serum was used for normalization (efficacy=1).
RESULTS AND DISCUSSION
The KYL, APY and VTM peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity
A previous phage display screen identified the KYL, APY and VTM peptides on the basis of their ability to bind to the extracellular portion of EphA4 . However, whether the peptides bind to the high-affinity ephrin-binding pocket of EphA4 was not conclusively demonstrated. We therefore performed isothermal titration calorimetry experiments with the ephrin-binding domain of human EphA4, which yielded KD values of 0.85±0.15 μM for KYL, 1.5±0.5 μM for APY and 4.7±0.1 μM for VTM (Figure 1). This confirms that all three peptides target the ephrin-binding domain of EphA4 and bind with substantial affinity. Interestingly the interaction of KYL, which has the highest-binding affinity, with EphA4 appears to involve a fast component followed by a slower (~400 s) component (Figure 1). This probably implies a slow conformational change in the receptor induced by KYL binding and would be consistent with the binding of the peptide to a particular conformation of the EphA4 ephrin-binding domain followed by re-equilibration of the different unbound receptor conformations [41,42]. In several Eph receptors the regions that form the sides of the ephrin-binding pocket (D, E, J and K β-strands and intervening loops) can modify their positions, accommodating different ligands [35–37,49,50]. This is particularly evident in EphA4, which is highly promiscuous and can bind both ephrin-A and -B ligands [35–37,41].
Residues in the ephrin-binding pocket of EphA4 that are important for the binding of the ephrin-A5 ligand
To examine whether residues within the EphA4 pocket that binds the natural ephrin ligands are also involved in binding the peptide ligands, and to identify specific amino acids that may play an important role in the interactions, we mutated residues in the ephrin-binding domain of EphA4 fused to AP (EphA4 AP) (Table 1). First, we examined the effects of the mutations on ephrin binding. ELISA assays measuring binding of the EphA4 AP mutants to the immobilized ephrin-A5 Fc ligand revealed that the T76A, F126A, I131A and R134A mutations severely impaired the EphA4–ephrin-A5 interaction (Table 1 and Supplementary Figure S1 at http://www.BiochemJ.org/bj/445/bj4450047add.htm; numbering of the residues is according to the construct used , where Asn29 in GenBank® accession number NP_004429 is the first residue). This confirms the critical importance of the four residues in ephrin binding, which was suggested by crystal structures showing their contact with bound ephrins [35,36]. The I31A, M32A, D33A, Q43A, D123A, M136A and A165S mutations also affect ephrin-A5 binding, which is also consistent with the previous structural studies suggesting the involvement of these residues, or the corresponding residues in other Eph receptors, in ephrin binding [35,51]. In contrast, the remaining mutations (S30A, I39A, T41A, V129A and G132V) do not substantially affect the interaction of EphA4 with ephrin-A5, suggesting that these residues are less critical for ephrin-A5 binding.
Residues in the ephrin-binding pocket of EphA4 are differentially involved in the binding of the KYL, APY and VTM peptide ligands
To obtain information on the effect of the mutations on peptide ligand binding, we examined the binding of the EphA4 AP mutants to the biotinylated peptides immobilized on ELISA wells (Table 1 and Supplementary Figure S2 at http://www.BiochemJ.org/bj/445/bj4450047add.htm). Interestingly, of the four amino acid changes that severely disrupt ephrin-A5 binding, only R134A also impaired binding of all three peptides, although the effect was weak in the case of APY. The F126A mutation substantially impaired KYL and VTM, but not APY, binding. The I131A mutation severely impaired VTM binding, KYL binding less severely and did not affect APY binding. Interestingly the fourth mutation, T76A, did not affect KYL binding, but increased APY binding and, more dramatically, VTM binding. On the other hand the T41A mutations, which did not affect ephrin-A5 binding, strongly impaired binding of all three peptides. These results reveal substantial differences in the EphA4 residues utilized for binding ephrin-A5 and the peptides.
The T41A, Q43A and A165A mutations strongly impair the binding of all three peptides, suggesting some common features in their interactions with EphA4. Additionally, however, three mutations (S30A, I31A and I39A) similarly affect the binding of KYL and APY, but not VTM, and four other mutations (M32A, F126A, G132V and R134A) similarly affect the binding of KYL and VTM, but not APY (Table 1). Thus KYL shares interacting residues in EphA4 with both APY and VTM, whereas APY and VTM bind most differently from each other. A complementary assay provided additional, albeit indirect, information on the effects of the mutations on the interactions between EphA4 and the peptides. This involved ELISAs measuring the ability of the peptides to antagonize EphA4–ephrin-A5 binding, which we used to evaluate those EphA4 AP mutants that retain substantial ability to interact with ephrin-A5 (Figure 2 and Supplementary Figure S3 at http://www.BiochemJ.org/bj/445/bj4450047add.htm). The effects of the EphA4 mutations on peptide antagonistic activity were mostly consistent with the effects on peptide binding, with a few exceptions. For example, although the APY peptide binds to the G132V EphA4 AP mutant as well as to wild-type, it did not inhibit ephrin binding to the mutant receptor. On the other hand, the VTM peptide does not bind to the A165S EphA4 AP mutant, but can nevertheless inhibit ephrin binding to the mutant receptor. These discrepancies may be explained by differential binding of the peptides to the EphA4 mutant conformation that binds the ephrin compared with the mutant conformations that predominate in the absence of ephrin . In addition, the KYL and VTM peptides showed impaired binding to the EphA4 M32A and D33A mutants, but they inhibited ephrin-A5 binding to the mutant receptors better than to the wild-type. This is probably due to the fact that the mutations also weaken ephrin binding. Overall, these results suggest that some residues in the EphA4 ephrin-binding pocket are important for the binding of all three peptides, whereas others are critical for the binding of only one or two of the peptides. Furthermore, the three peptide ligands do not closely mimic the binding of the ephrin-A5 ligand.
To obtain a more complete overview of the residues in the EphA4 ephrin-binding domain that are involved in KYL binding, we compared the NMR HSQC spectra of EphA4 alone or in complex with GS-KYL, a modified version of KYL containing N-terminal glycine and serine residues derived from the bacterial expression construct. The CSDs (chemical-shift differences) identified a number of EphA4 residues that are affected by KYL binding (Figure 3 and Table 1), which may be caused by a direct interaction or conformational/dynamic changes occurring as a result of peptide binding [36,41]. All of the residues with high CSD values (>0.4) are located within or near the ephrin-binding pocket of EphA4 [35,51]. They include Ile31, Met32, Asp33, Thr41, Gln43, Phe126, Ile131, Gly132 and Ala165 which are all shown to be important for KYL binding in our mutagenesis experiments. Interestingly, although the chemical shift differences for Ser30 and Thr76 are very large (Figure 3B) and the two residues have close contacts with the KYL peptide in a model of the complex (Figure 5B), mutation of these residues to alanine does not affect KYL binding (Supplementary Figure S2). This implies that the introduced alanine residue may not be sufficiently different from serine or threonine to substantially affect KYL binding or that the energetic contribution of the two residues to the binding of the peptide may be minor, given that the magnitude of the CSD and the contribution of a residue to the binding energy are not always correlated .
Residues of KYL and VTM that are critical for interaction with EphA4
To identify the residues of the KYL and VTM peptides that are important for binding to EphA4, we replaced each of their amino acids with alanine, except for the two alanine residues in VTM that were replaced with serine. We did not perform a similar analysis for APY, in which the two cysteine residues probably form a disulfide bond that cyclizes the peptide and is critical for its binding activity. It was found that seven of the 12 amino acids in KYL (Lys1, Tyr2, Trp6, Pro7, Val8, Leu9 and Leu12) and eight of the 12 amino acids in VTM (Thr2, Met3, Glu4, Iso6, Asp7, Leu8, Phe10 and Pro11) are essential for high-affinity binding because substitution of each of these amino acids with alanine severely impaired the ability of the peptides to inhibit the EphA4–ephrinA5 interaction in ELISAs (Figure 4). The other residues play a lesser role or do not appear to play a role in EphA4 binding. For example, replacement of Leu3, Pro4 and Tyr5 with alanine decreased the inhibitory activity of KYL in ELISAs by only 2–4-fold, whereas replacement of Ser10 and Ser11 resulted in an IC50 value that is comparable with that of unmodified KYL. Furthermore, replacement of Val1, Ala5 and Ala9 reduced the inhibitory potency of VTM by only 2–4-fold, and replacement of Gly12 by less than 2-fold (Figure 4).
Model of the EphA4–KYL complex
We have attempted to determine the three-dimensional structure of the EphA4–KYL complex by both crystallography and NMR spectroscopy. However, attempts to co-crystallize the EphA4 ephrin-binding domain in complex with the synthetic KYL peptide led to several crystals of EphA4 without KYL. On the other hand, NMR structure determination was hindered by the extensive disappearance of NMR resonance signals for EphA4 side chains in the presence of the recombinant GS-KYL peptide. Therefore we used the PDB 3CKH EphA4 structure for molecular docking of the GS-KYL peptide with the HADDOCK software using the information obtained from the NMR chemical shifts, EphA4 mutagenesis and alanine scanning outlined above. In the model, the KYL peptide is buried in the EphA4 ephrin-binding pocket (Figure 5A). The EphA4 residues with significant NMR perturbations and critical for binding as revealed by mutagenesis were all located in the pocket accommodating the KYL peptide (Figure 5B). The KYL peptide is characterized by a positively charged N-terminus and a rather hydrophobic middle region. In the model of the complex, side chain amide protons of KYL residue K1 form two hydrogen bonds with EphA4 Glu27, one with the backbone and another with side chain oxygen atoms. The aromatic ring of KYL Tyr2 is in close contact with EphA4 Leu83, a residue that undergoes a large shift upon KYL binding (Figure 3B). Hydrophobic KYL residues Trp6, Pro7, Val8 and Leu9 establish extensive contacts with the hydrophobic patches of the ephrin-binding pocket of EphA4 (Figure 5A). For example, Trp6 is involved in hydrophobic contacts with EphA4 residues Phe126, Val129, Ile131 and Ala165, in agreement with the mutagenesis and NMR titration results. Additionally, the backbone amide proton of Trp6 forms a hydrogen bond with the side chain oxygen of EphA4 Thr76, consistent with the NMR titration data, indicating that Thr76 was significantly perturbed. However, the EphA4 T76A mutation did not substantially affect KYL-binding affinity (Table 1), suggesting that Thr76 may make a very minor energetic contribution to KYL binding. This would not be surprising because a portion of interfacial residues in protein complexes can have a minor, or even negative, energetic contribution to the formation of complexes . Pro7 appears to be particularly important for KYL binding because, in addition to making direct contacts with EphA4 Ser30 and Thr41, it may induce and stabilize a bend in the backbone of the peptide that allows residues Val8 and Leu9 to form hydrophobic contacts with EphA4 residues Thr41 and Ile131 respectively. This is consistent with the complete loss of binding of the P7A-modified KYL (Figures 4A–4C). Leu12 is also essential for binding because its hydrophobic side chain bends to form a hydrophobic cluster with the side chains of Pro7 and Leu9 from KYL, thus stabilizing their orientation, which is required for interaction with EphA4 residues. Moreover, the amide proton of Leu12 forms a hydrogen bond with the backbone oxygen of Gly132.
Use of EphA4-binding peptides for receptor purification and cell imaging
Given their substantial binding affinity, antagonistic properties and selectivity for EphA4 , the three peptides represent useful tools for a number of applications. Although KYL has already been used to modulate EphA4 function in various biological systems [6,13,38,43,44], we also found that binding of all three peptides to EphA4 was sufficiently stable to enable pull down of the receptor from cell and tissue lysates (Figures 6A and 6B). Thus these peptides may be useful to purify EphA4 protein or isolate tumour cells expressing high levels of EphA4 [53–55]. Furthermore, KYL coupled to fluorescent quantum dots can be used to image cells expressing EphA4 (Figure 6C). Thus KYL coupled to fluorescent or radioactive tags may serve to image EphA4-expressing tumours in vivo, similar to other Eph receptor-targeting peptides [56,57].
Stability of KYL, APY and VTM in cell culture medium and mouse serum
To evaluate the stability of the peptides for use in cell culture experiments, we measured their ability to inhibit the EphA4–ephrin-A5 interaction in ELISAs after different incubation times in culture medium conditioned by human PC3 prostate cancer cells or mouse C2C12 myoblasts cells. The KYL and APY peptides appeared to be quite stable in cell culture medium, with a half-life of ~8–12 h in medium conditioned by PC3 and C2C12 cells (Figure 7). The VTM peptide was even more stable, with a half-life of ~30 and 90 h respectively. On the other hand, the antagonistic activity of the peptides was lost within 10–40 min of incubation in mouse serum, with VTM again being more stable than KYL and APY (Figure 7). Thus modifications that increase the half-life of the peptides in the blood circulation will be useful for in vivo applications, similar to what we have observed for a peptide that targets the EphB4 receptor .
In conclusion, we have characterized three peptides that selectively bind to the EphA4 receptor and identified features that are important for peptide–receptor interaction. We have generated a series of mutations in the EphA4 ephrin-binding pocket and found that many of them differentially affect the binding of the three peptide ligands as well as the natural ephrin-A5 ligand to EphA4, suggesting that each ligand interacts in a distinctive manner with residues of the high-affinity ephrin-binding pocket of EphA4, perhaps by binding to different conformations of the receptor . The peptides target the high-affinity ephrin-binding pocket of EphA4, which is used by the G–H loop of all the ephrins in a promiscuous manner. In contrast, the peptides are highly selective for EphA4  and therefore probably exploit a unique feature of the receptor's pocket. Furthermore, the alanine scans of the KYL and VTM peptides identify amino acids that are essential for the inhibitory properties of the peptides. They also reveal that five amino acids in KYL and four in VTM do not substantially contribute to the antagonistic ability of the peptides and could therefore be modified to obtain a more powerful inhibitor or increase peptide stability. The first and the last amino acids of VTM, but not KYL, appear to be dispensable for high-affinity binding and therefore the VTM peptide may be shortened to 10 amino acids without appreciable loss of binding affinity. In future applications, KYL, APY and VTM, as well as optimized derivatives, could be conjugated with drugs, toxins and imaging agents or incorporated into nanoparticles to selectively target cells with high EphA4 levels.
Ilaria Lamberto generated most of the EphA4 mutants and carried out the ELISA experiments; Roberta Noberini generated the EphA4 Q43A mutant and carried out initial ELISA experiments; Haina Qin performed the NMR experiments and computer modelling; Lakshmanane Premkumar generated EphA4 protein for the isothermal titration calorimetry experiments; Caroline Bourgin performed the peptide pull-down experiments; Jianxing Song, Stefan Riedl and Elena Pasquale helped with design and interpretation of the experiments and supervised the work; Ilaria Lamberto and Elena Pasquale wrote the paper with help from the other authors.
This work was supported by the National Institutes of Health [grant numbers CA138390 (to E.B.P. and S.R.) and NS067502 (to E.B.P.)] and the Singapore National Medical Research Council [grant number NMRC/1216/2009 (to J.S.)].
The authors thank A. Bobkov for performing the isothermal titration calorimetry experiments, G. Scicolone for the EphA4–EGFP construct, and L.D. Nguyen for staining cells with KYL peptide bound to quantum dots.
Abbreviations: AP, alkaline phosphatase; CSD, chemical-shift difference; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; EGFP, enhanced green fluorescent protein; FBS, fetal bovine serum; HADDOCK, high-ambiguity-driven protein–protein docking; HSQC, heteronuclear single-quantum coherence; TBS, Tris-buffered saline
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