Structural Characterization of the EphA4-Ephrin-B2 Complex Reveals New Features Enabling Eph-Ephrin Binding Promiscuity

Abstract

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 Gln 12 and Glu 14 and ephrin-B2. Mutation of Gln 12 and Glu 14 does not cause significant structural changes in EphA4 or changes in its affinity for ephrin-A ligands. However, the EphA4 mutant has ϳ10-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 Gln 12 and Glu 14 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.The 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 (1, 2).All of the Eph receptors share the same modular structure, which comprises a juxtamembrane region, a tyrosine kinase domain, a C-terminal sterile ␣-motif domain, and a PDZbinding 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 ␤-strands organized in a jellyroll ␤-sandwich architecture, which is conserved among EphA and EphB receptors (3-8). The ectodomain of the ephrins is also conserved and consists of an eight-stranded ␤-barrel with a Greek key topology, including several large and highly conserved functional loops, such as the G-H and C-D loops (4,5,8,9), which are very flexible in solution (10).The formation of a complex between an Eph receptor and an ephrin is centered around the insertion of the solventexposed ephrin G-H loop into the Eph receptor hydrophobic channel formed by the convex sheet of four ␤-strands together with the D-E, J-K, and G-H loops. These interactions are...

Insights into Eph receptor tyrosine kinase activation from crystal structures of the EphA4 ectodomain and its complex with ephrin-A5

“…As mentioned above, both of these regions are integral parts of the clustering interface in the ligand/receptor complex. The same ligand-induced conformational change in the EphA4 LBD was also observed in the studies reporting the structures of the EphA4-LBD and the EphA4-LBD/ephrin-A2 and ephrin-B2 complexes (8,16,18), but because the isolated Eph LBD does not cluster, its significance was not appreciated.…”

“…The ligand-binding cavity of the receptor consists of loops DE, FG, and JK, which were shown to exhibit various amounts of conformational flexibility, depending on the subclass and the individual receptor. Several structures of the EphA4 ligand-binding domain (LBD) alone and in complex with its ligands have addressed its unusually high ligand promiscuity (8,(16)(17)(18).More recently, the crystal structures of the EphA2 ectodomain (ECD) bound to ephrin-A1 and ephrin-A5 suggested a structural basis for the formation of Eph/ephrin signaling clusters at the interfaces of interacting cells (19,20). Specifically, it was proposed that these signaling clusters nucleate from high-affinity Eph/ephrin heterodimers, which assemble into heterotetramers that further aggregate into higher-order oligomers.…”

“…The ligand-binding cavity of the receptor consists of loops DE, FG, and JK, which were shown to exhibit various amounts of conformational flexibility, depending on the subclass and the individual receptor. Several structures of the EphA4 ligand-binding domain (LBD) alone and in complex with its ligands have addressed its unusually high ligand promiscuity (8,(16)(17)(18).…”

Blockade of EphA4 signaling ameliorates hippocampal synaptic dysfunctions in mouse models of Alzheimer’s disease

“…Accordingly, it would be of great interest to study whether blocking the EphA4 activity is a promising intervention strategy for AD. (31,33). Therefore, a virtual screening of an in-house traditional Chinese medicine (C and D); or CA1 regions of WT and APP/PS1 mutant mice were injected with EphA4-shRNA (shEphA4) or GFP virus (E and F).…”

EphB Receptors Trigger Akt Activation and Suppress Fas Receptor-Induced Apoptosis in Malignant T Lymphocytes

“…In mammals, EphA receptors (EphA1-8, EphA10) promiscuously bind ephrin-A proteins (ephrin-A1-6), and EphB receptors (EphB1-4, EphB6) interact in a similar manner with three ephrin-Bs (ephrin-B1-3) (31). This rule, however, is not without exceptions, because EphA4 is capable of binding ephrin-Bs, and EphB2 can be activated by ephrin-A5 in addition to their conventional ephrin-A and ephrin-B ligands (32)(33)(34)(35)(36). In contrast, EphB6 interactions appear to be restricted to ephrin-B1 and ephrin-B2 (34,37,38).…”