Figure 1.
The crystal structure of the EphA5 LBD shows well defined D–E and J–K loops.
(a) Crystal structure of the EphA5 LBD. Residues Ala179-Ser182 and Gly189-M193 in the J–K loop, which adopt unusual helical-like conformations, are displayed in red. Two disulfide bridges Cys137-Cys147 and Cys102-Cys220, are displayed in orange. (b, c) Electron density maps for the D–E and J–K loops, showing that all residues are well defined.
Figure 2.
The structure of the unbound EphA5 LBD resembles that of other Eph receptors bound to ephrin ligands.
(a) Superimposition of the LBD structures of unbound EphA5 (red), EphA2 (green, 3C8X), EphA4 (yellow, 3CKH) and EphB2 (blue, 3ETP). A short β-sheet is formed by the EphA4 and EphB2 residues corresponding to EphA5 residues Ala179-Ser182 and Gly189-M193 in the J–K loop (orange arrows). (b) Superimposition of the LBD structures of the unbound EphA5 (red), EphA2 in complex with ephrin-A2 (green, 3CZU), EphA4 with ephrin-A2 (cyan, 3WO3), EphA4 with ephrin-B2 (blue, 3GXU), EphB2 with ephrin-B2 (pink, 1KGY) and EphB4 with ephrin-B2 (yellow, 2HLE).
Figure 3.
Unique ligand-binding specificity of the EphA5 LBD.
(a) Isothermal titration calorimetry profiles for the interaction of the EphA5 LBD with the WDC peptide (upper panel) and plots of the integrated values for the reaction heats (after blank subtraction and normalization to the amount of the peptide injected) versus EphA5 to WDC molar ratio (lower panel). The thermodynamic binding parameters are shown in the lower panel. (b) Inhibition of ephrin-A5 alkaline phosphatase (AP) binding to immobilized EphA5 Fc by increasing concentrations of WDC in ELISAs. Bound ephrin-A5 AP represents the ratio of the OD at 405 nm for ephrin-A5 AP bound to EphA5 Fc in the presence of the indicated concentrations of the WDC peptide and in the absence of peptide. (c) Inhibition of ephrin-A5 AP binding to EphA receptors and ephrin-B2 AP binding to EphB receptors by 100 μM WDC. Bound ephrin AP represents the ratio of the OD at 405 nm for ephrin-A5 AP or ephrin-B2 AP bound to different Eph receptor Fc proteins in the presence of WDC peptide and in the absence of peptide. The peptide substantially inhibits ephrin binding only to EphA5. Averages and standard errors from triplicate measurements are shown. (d) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of WDC at a molar ratio of 1:3 (EphA5:WDC). (e) Superimposition of the NMR HSQC spectra of the EphA5 LBD in the absence (blue) and in the presence (red) of C1 at a molar ratio of 1:20 (EphA5:C1).
Figure 4.
Structural properties and solvent accessibility of the EphA5 LBD in solution.
(a) Residue-specific Cα chemical shift deviations (ΔCα = δobs – δcoil) for the EphA5 LBD. The bars for the J–K loop residues with helical conformations in the crystal structure are colored in red. (b) Superimposition of 1H-15N NMR HSQC spectra for the 15N-labeled EphA5 LBD at 25°C in 10 mM phosphate buffer, pH 6.5 (blue) and 15 min after dissolving the lyophilized sample in D2O (red). The disappearance of the blue HSQC peaks indicates the high exposure of the amide protons to the solvent. (c) Superimposition of HSQC spectra of the EphA5 LBD at 25°C, 15 min (blue) and 24 hours (red) after dissolving the lyophilized sample in D2O. (d) EphA5 LBD structure with residues whose HSQC peaks are missing even in H2O buffer colored in green, residues whose backbone amide protons completely exchanged within 15 min in blue, residues whose backbone amide protons persisted after 15 min but completely exchanged in 2 hours in yellow, and residues whose backbone amide protons persisted even after 2 hours in red. The very rapid exchange of their amide protons indicates that the D and E strands are highly exposed to the solvent.
Figure 5.
15N backbone dynamics for the EphA5 LBD on the ps-ns time scale.
(a) Generalized squared order parameter (S2) derived from the Model-free analysis of the relaxation data for EphA5. Red indicates residues with S2 < the average value. (b) Residue-specific Rex derived from Model-free analysis of relaxation data for EphA5 (green) and EphA4 (red and light brown). Red indicates EphA4 residues in the D and E strands as well as D–E and J–K loops while light brown for the other EphA4 residues. (c) EphA5 LBD structure with residues having S2 < the average value (0.7) colored in green and those with S2 < the average – STD (0.5) in red. (d) EphA5 LBD structure with residues having Rex >2 Hz colored in cyan and those >5 Hz colored in red.
Figure 6.
Distinctive dynamic behaviors of the EphA5 LBD as revealed by molecular dynamics simulations.
(a) Trajectories of root-mean-square deviations (RMSD) of heavy atoms in three independent molecular dynamics simulations. (b) Trajectories of root-mean-square fluctuations (RMSF) of the Cα atoms computed for three independent simulations, with average values and standard deviations calculated over 30 ns for each simulation. (c) EphA5 LBD structure with the residues having RMSF >average in green and those >2-fold the average in red.
Figure 7.
Molecular dynamics simulations reveal that the unbound EphA5 ephrin-binding pocket is in an open conformation.
(a–c) Superimposition of structure snapshots taken at 1 ns intervals in three independent molecular dynamics simulations. (d) Trajectories of the distances between the Cα atoms of Glu91 in the D–E loop and Val192 in the J–K loop over 30 ns simulations. The average values and standard deviations calculated over the 30 ns are displayed for each simulation. The EphA5 ligand binding pocket still remains open even after 30 ns in all three simulations.
Figure 8.
Sequence-structure relationship for the EphA5 and EphA4 LBDs.
(a) Alignment of the sequences of the EphA5 and EphA4 LBDs. Identical residues are colored in blue, homologous in green and different in black. Residues in the D and E β-strands are highlighted in yellow and residues in the J–K loop in pink. Two residues that are in close contact in the EphA4 structure (Ile in the D strand and Asp in the J–K-loop) and the corresponding residues in the EphA5 LBD are boxed. (b) Structure of the EphA5 LBD with spheres for Asp190 in the J–K-loop, and Glu80 in the D strand which corresponds to a Ile in the structure of the EphA4 LBD (c).