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. 2008 Oct 24;283(43):29473-84.
doi: 10.1074/jbc.M804114200. Epub 2008 Aug 14.

Crystal structure and NMR binding reveal that two small molecule antagonists target the high affinity ephrin-binding channel of the EphA4 receptor

Haina Qin  1 , Jiahai Shi, Roberta Noberini, Elena B Pasquale, Jianxing Song
Affiliations

Crystal structure and NMR binding reveal that two small molecule antagonists target the high affinity ephrin-binding channel of the EphA4 receptor

Haina Qin et al. J Biol Chem. .

Abstract

The Eph receptor tyrosine kinases regulate a variety of physiological and pathological processes not only during development but also in adult organs, and therefore they represent a promising class of drug targets. The EphA4 receptor plays important roles in the inhibition of the regeneration of injured axons, synaptic plasticity, platelet aggregation, and likely in certain types of cancer. Here we report the first crystal structure of the EphA4 ligand-binding domain, which adopts the same jellyroll beta-sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core beta-stranded regions, whereas large variations exist in the loops, particularly the D-E and J-K loops, which form the high affinity ephrin binding channel. We also used isothermal titration calorimetry, NMR spectroscopy, and computational docking to characterize the binding to EphA4 of two small molecules, 4- and 5-(2,5 dimethyl-pyrrol-1-yl)-2-hydroxybenzoic acid which antagonize ephrin-induced effects in EphA4-expressing cells. We show that the two molecules bind to the EphA4 ligand-binding domain with K(d) values of 20.4 and 26.4 microm, respectively. NMR heteronuclear single quantum coherence titrations revealed that upon binding, both molecules significantly perturb EphA4 residues Ile(31)-Met(32) in the D-E loop, Gln(43) in the E beta-strand, and Ile(131)-Gly(132) in the J-K loop. Molecular docking shows that they can occupy a cavity in the high affinity ephrin binding channel of EphA4 in a similar manner, by interacting mainly with the EphA4 residues in the E strand and D-E and J-K loops. However, many of the interactions observed in Eph receptor-ephrin complexes are absent, which is consistent with the small size of the two molecules and may account for their relatively weak binding affinity. Thus, our studies provide the first published structure of the ligand-binding domain of an EphA receptor of the A subclass. Furthermore, the results demonstrate that the high affinity ephrin binding channel of the Eph receptors is amenable to targeting with small molecule antagonists and suggest avenues for further optimization.

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Figures

FIGURE 1.
FIGURE 1.
Crystal structure of the EphA4 ligand-binding domain. a, stereo view of the two disulfide bridges in the EphA4 ligand-binding domain built into the simulated-annealing 2Fo - Fc electron density map contoured at 1. 5σ. b, ribbon representation of two EphA4 ligand-binding domain molecules A and B (Mol-A and Mol-B) in one asymmetric unit. The red arrows are used to indicate the novel interface between the two molecules. c, ribbon representation of two EphA4 molecules in one asymmetric unit that have differential packing contacts with molecules in other asymmetric units.
FIGURE 2.
FIGURE 2.
Structural comparison. a, stereo view of the superimposition of the two EphA4 ligand-binding domain structures observed in the same asymmetric unit (structure A is in purple and structure B is in green). b, stereo view of the superimposition of two EphA4 structures (structure A in red and structure B in lime green) with previously determined EphB2 and EphB4 structures (all in purple).
FIGURE 3.
FIGURE 3.
Characterization of the interactions with two small molecule antagonists. a, far-UV circular dichroism spectra of the EphA4 ligand-binding domain in the absence (black) and in the presence of compound 1 (blue) or compound 2 (red). The chemical structures of the two compounds are presented. b, 1H-15N NMR HSQC spectra of the EphA4 ligand-binding domain in the absence (blue) and in the presence of compound 1 (red). c, residue-specific CSD of the EphA4 ligand-binding domain in the presence of compound 1. d, residue-specific CSD of the EphA4 ligand-binding domain in the presence of compound 2. Violet bars indicate residues with CSD larger than 2.5 times of the standard deviation as described under “Experimental Procedures.” In all experiments the molar ratio of EphA4 to compound was 1:6.
FIGURE 4.
FIGURE 4.
Models of structure A in complex with small molecule antagonists. a, stereo view of the superimposition of the unbound EphA4 structure A (green) with its three selected docking models in complex with compound 1 (brown). b, stereo view of the superimposition of the unbound EphA4 structure A (green) with its three selected docking models in complex with compound 2 (brown). Both sticks and dots are used to highlight residues Ile31-Met32 in the D-E loop, Gln43 in the E β-strand, and Ile131-Gly132 in the J-K loop.
FIGURE 5.
FIGURE 5.
Models of structure B in complex with small molecule antagonists. a, stereo view of the superimposition of the unbound EphA4 structure B (green) with its three selected docking models in complex with compound 1 (brown). b, stereo view of the superimposition of the unbound EphA4 structure B (green) with its three selected docking models in complex with compound 2 (brown). Both sticks and dots are used to highlight residues Ile31-Met32 in the D-E loop, Gln43 in the E β-strand, and Ile131-Gly132 in the J-K loop.
FIGURE 6.
FIGURE 6.
EphA4 binding cavity for the small molecule antagonists. Surface representation of the EphA4 binding cavity of the docking model with the lowest energy. a, EphA4 structure A with compound 1; b, EphA4 structure A with compound 2; c, EphA4 structure B with compound 1; d, EphA4 structure B with compound 2. The small molecule antagonists are represented by sticks and oxygen atoms are colored in red. EphA4 residues Ile31-Met32 in the D-E loop are in brown; residue Gln43 in the E β-strand is in blue/purple, and residues Ile131-Gly132 in the J-K loop are in violet.
FIGURE 7.
FIGURE 7.
Comparison of the EphA4 small molecule models with EphB receptors in complex with ephrins or peptides. a, stereo view of the superimposition of four selected EphA4 small molecule models with previously determined structures of EphB-ephrin complexes (PDB codes 1KGY, 1SHW, and 2HLE). EphA4 is represented by a yellow ribbon and the small molecules by green dots. The EphB receptors are in purple, and ephrin-B2/ephrin-A5 are in red. The blue arrows indicate the contact regions outside of the ligand binding channel that contribute to the high affinity Eph receptor-ephrin binding interface. b, stereo view of the superimposition of four selected EphA4 small molecule models with previously determined structures of EphB-peptide complexes (PDB codes 2QBX and 2BBA). EphA4 is in yellow; EphB receptors are in purple; one peptide is in red and another in pink. The blue arrow indicates a conserved binding motif identified in all the EphB structures in complex with either ephrins or antagonistic peptides (see Ref. 15).
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