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 -sandwich architecture as shown previously for EphB2 and EphB4. The similarity with EphB receptors is high in the core -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. The erythropoietin-producing hepatocellular (Eph) 3 carcinoma receptors constitute the largest family of receptor tyrosine kinases, with 16 individual receptors throughout the animal kingdom, which are activated by nine ephrins (1-6). Eph receptors and their ligands are both anchored onto the plasma membrane and are subdivided into two subclasses (A and B) based on their sequence conservation and binding preferences. Usually, EphA receptors (EphA1-A10) interact with glycosylphosphatidylinositol-anchored ephrin-A ligands (ephrin-A1-A6), whereas EphB receptors (EphB1-B6) interact with transmembrane ephrin-B ligands (ephrin-B1-B3) that have a short cytoplasmic portion carrying both Src homology domain 2 and PDZ domain-binding motifs (7,8).The Eph receptors have a modular structure, consisting of a unique N-terminal ephrin-binding domain followed by a cysteine-rich linker and two fibronectin type III repeats in the extracellular region. The intracellular region is composed of a conserved tyrosine kinase domain, a C-terminal sterile ␣-domain, and a PDZ-binding motif. The N-terminal 180-residue globular domain of the Eph receptors has been shown to be sufficient for high affinity ephrin binding (9 -11). EphA subclass receptors remarkably differ from EphB receptors because they lack a 4-residue insert in the H-I loop of the ligand-binding domain. Previously, the structures of the EphB2 and EphB4 ligand-binding domains have been determined in both the free state and in complex with ephrins or peptide antagonists (10,11,(12)(13)(14)(15). These studies have shown that the ligand-binding domains of EphB2 and EphB4 adopt the same jellyroll -sandwich architecture composed of 11 antiparallel -strands connected by loops of various lengths. In particular, the D-E and * This work was supported by National Medical Research Council of Singapore Grant R-154-000-382-213 (to J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must the...