K_D Determination of EphA4 LBD with Ephrin RBD
(A–E) Overlay association plots of surface plasmon resonance sensorgrams recording response of EphA4 (analyte) interaction with immobilized ephrinA1 (A), ephrinA2 (B), ephrinA4 (C), ephrinA5 (D), and ephrinB2 (E). Calculated affinity binding K_D values are shown.

Structural Alignment of Ephrin Ligands in Their Bound States
Ephrin A class ligands (A) and ephrinB2 ligands (B) are shown in cartoon representation. EphrinA1 from EphA2-ephrinA1 (PDB code 3CZU) is colored green, ephrinA2 from EphA4-ephrinA2 is colored orange, and ephrinA5 from EphB2-ephrinA5 (PDB code 1SHW) is colored blue. EphrinB2 from EphA4-ephrinB2 is colored cyan, ephrinB2 from EphB2-ephrinB2 (PDB code 1KGY) is colored yellow, and ephrinB2 from EphB4-ephrinB2 (PDB code 2HLE) is colored gray.

EphrinB2 Precludes the Formation of an α Helix in the JK^EphA4 Loop upon Binding
(A) Residues from the GH^ephrinB2 loop (stick representation with nitrogen blue, oxygen red, and carbon green) bind between the DE^EphA4 and JK^EphA4 loops (stick representation with nitrogen blue, oxygen red, sulfur gold, and carbon gray).
(B) Residues from the GH^ephrinA2 loop (stick representation with nitrogen blue, oxygen red, and carbon yellow) bind between the DE^EphA4 and JK^EphA4 loops (gray van der Waals surface; stick representation with nitrogen blue, oxygen red, and carbon gray).
(C) W125^ephrinB2 from the GH^ephrinB2 loop (green sticks, colored as in [A]) clashes with M164^EphA4 when superposed onto EphA4 in its ephrinA2-bound state.

The Extended Ephrin Binding Site
(A–E) Eph and ephrin Cα traces where EphA4 is colored gray, ephrinA2 is colored pink, and ephrinB2 is colored cyan.
(A) Comparison of ephrinA2 (pink)- and ephrinB2 (cyan)-bound to EphA4 (gray) based on superposition of the EphA4 component (shown in stereo representation) reveals a 12° relative tilt between ephrinA- and ephrinB-bound complexes.
(B and C) The presence or absence of an extended Eph-ephrin binding surface correlates with the degree of van der Waals and hydrogen bonding interactions. Midpoints between van der Waals interactions are shown as gold surfaces and intracomplex hydrogen bonds are shown as red spheres in the EphA4-ephrinA2 (B) and EphA4-ephrinB2 (C) complexes. Boxes in (B) and (C) refer to areas highlighted in (D) and (E). Colored as in (A).
(D and E) Van der Waals and hydrogen bonding interactions taking place in the extended binding surface. Residues involved in protein-protein contacts are shown as sticks where carbon atoms are colored as in (A)–(C), nitrogen atoms are colored blue, oxygen red, and carbon yellow. Dashed lines correspond to intracomplex hydrogen bonds.

The Structure of the EphA4 LBD
(A) Cartoon representation of the apo LBD of EphA4 colored as a rainbow with the N terminus in blue and the C terminus in red and with secondary structure labeled according to standard nomenclature.
(B–D) Cα traces.
(B) Superposition of all EphA4 molecules from the asymmetric unit of reported unbound crystal structures. Molecules in the asymmetric unit of the EphA4 structure we report here are colored blue, and molecules in the asymmetric unit of PDB code 3CKH are colored orange.
(C) Representative EphA4 molecule colored according to regions of flexibility as defined by ESSET (Schneider, 2002). Regions colored blue are conformationally invariant and regions colored red are flexible.
(D) Superposition of representative EphA4 (light blue) with crystal structure of unbound EphA2 (green; PDB code 3C8X).

Crystal Structures of EphA4-ephrinB2 and EphA4-ephrinA2
(A and B) Cartoon representation of EphA4-ephrinB2 (A) and EphA4-ephrinA2 (B). Secondary structure elements are labeled according to standard nomenclature in A and the main interaction loops are labeled in B. The JK^Eph loops are marked with an asterisk and undergo major conformational changes dependent on the class of ephrin ligand bound. Acetyl-glucosamine moieties observed at N-linked glycosylation sites are shown as pink sticks.

Conformational Changes of EphA4 upon Binding
(A) Superposition of unbound EphA4 (orange), with its ephrinB2 (dark blue)- and ephrinA2 (gray)-bound states shown in stereo representation.
(B) Rms displacement of equivalent residues between unbound, ephrinB2-bound, and ephrinA2-bound EphA4 mapped onto the Cα trace structure of EphA4 receptor (from EphA4-ephrinA2). The tube radius and color of the trace represent the rms displacement (ramped from blue to red). Regions with high deviations between structures are thick and colored red, while regions with low deviation are thin and colored blue.
(C) Cα trace of EphA4 from EphA4-ephrinA2 colored according to regions of flexibility as defined by ESSET (Schneider, 2002). Regions colored blue are conformationally invariant and regions colored red are flexible.

Structure and Sequence Conservation of EphA-ephrinA and EphB-ephrinB Complexes
(A) Superposition of EphA4 (yellow) and EphA2 (cyan) from their ephrinA2- and ephrinA1-bound states, respectively.
(B) Superposition of EphB2 (brown; PDB code 1KGY) and EphB4 (magenta; PDB code 2HLE) from their ephrinB2-bound states.
(C and D) EphA4 from its ephrinA2-bound state (C; yellow) and EphB2 from its ephrinB2-bound state (D; brown; PDB code 1KGY) with sequence conservation mapped onto the structure as blue (residues completely conserved between EphA sequences) and green (residues at least 80% conserved across Eph sequences) spheres.
(E) Sequence alignment of the DE^Eph loop numbered according to EphA4.
(F) Sequence alignment of the JK^Eph loop numbered according to EphA4.

Structure-Based Phylogenetic Analysis of Eph Receptors and Ephrin Ligands
(A and B) Unrooted trees of the Eph receptor LBD (A) and ephrin ligand RBD (B). The structures were superposed and a pairwise distance matrix was constructed as previously described (Graham et al., 2008). PDB codes for each representative structure are labeled beneath a cartoon representation ramped from blue (N terminus) to red (C terminus). Branches colored blue correspond to A class Eph and ephrin families, branches colored red correspond to B class Eph and ephrin families, and the branch colored lilac (A) corresponds to EphA4 from its ephrinB2-bound state, which does not belong to either family. Structures marked with an asterisk are those presented within this manuscript.