EphA2/ephrin-A1 (5) structures, structural alignment, and ligand binding. (A) Left, backbone representation of superimposed structures. Right, all structures are shown from the same perspective in ribbon format. Each structure is labeled according to its PDB code and differentially colored. (B) The EphA2-EphrinA1 high-affinity heterodimer interface. Stereoscopic view of the interface with domains labeled and shown in ribbon format. Ligand residues that are within 4 Å of the LBD are shown as sticks; LBD residues as lines. Water molecules in the vicinity are shown as red spheres, hydrogen bonds as black dashed lines.

Eph/ephrin heterotetramerization assembly. (A) Ribbon and (B) surface representations of the LBD-ephrin-A5 heterotetramer with each domain colored differently. (C) Detailed stereoscopic view of the 2∶2 assembly.

Cellular studies. (A) Immunoprecipitates from HEK293 cells transfected with increasing amounts of GFP-tagged WT EphA2, ΔLBD-EphA2, or ΔCRD-EphA2 were immunoblotted with α-EphA2 and α-phosphotyrosine antibodies. Densitometry quantified EphA2 phosphorylation relative to EphA2 expression, by using samples with most similar EphA2 levels (lanes 2, 5, and 8). (B) Activation of WT and mutated EphA2 in HEK293 cells by preclustered ephrin-A5. Single = L223R, double = L223R,L254R, and triple = L223R,L254R,V255R. (C) HEK293 cells stably expressing WT or point mutated EphA2, or control cells, were transfected with ΔLBD-EphA2-GFP (ΔLBD) and stimulated with clustered ephrinA5-Fc. Protein A sepharose pull-downs of ephrinA5-Fc associated receptors were Western blotted with α-EphA2 and α-GFP antibodies. The graph shows the amount of ΔLBD pulled down (anti-GFP blot) via association with full-length EphA2, relative to full-length EphA2, quantified by densitometry. (D) Parental HEK293 cells or derived clones stably expressing WT EphA2 or EphA2 point mutants as indicated were transfected with ΔLBD-EphA2-GFP. Alexa594-ephrinA5 conjugated Dynabeads were added to the cells for 5 min before cultures were rinsed and fixed for microscopy. Panels show representative images of EphA2-GFP and Alexa594-ephrinA5 fluorescence, white arrows indicating beads with recruited EphA2-GFP, yellow arrows indicating beads in contact with cells lacking recruitment. Insets show higher magnification images of boxed regions. The average proportion of beads in contact with cells that recruited ΔLBD-EphA2-GFP was determined for each cell line (WT EphA2, triple or double mutant or control cells) and is shown in the graph (± SEM).

Eph/ephrin assemblies. Left, ribbon diagram of four molecules each of receptor and ligand are shown with receptors colored differently for clarity. Right, two assemblies are shown; the first (“heterotetramerization assembly”) is mediated only by the LBD, the second (“clustering assembly”) is mediated by both LBD and CRD. This figure was generated from the EphA2/ephrin A5 complex but also applies to the other, ephrin-A1, complex structures.

Clustering assembly. (A) Ribbon display of the clustering assembly with each EphA2 molecule colored differently; (B) detailed stereoscopic view of the LBD-mediated clustering interaction. Middle, ribbon diagram of two molecules of the unbound EphA2—dimer in light orange and pale green. (C) Detailed view of CRD-mediated clustering. Residues that mediate clustering are shown in stick format and labeled. Dashes represent van der Waals or hydrogen bond interactions.