Protein dynamics at Eph receptor-ligand interfaces as revealed by crystallography, NMR and MD simulations

Abstract

Background: The role of dynamics in protein functions including signal transduction is just starting to be deciphered. Eph receptors with 16 members divided into A- and B- subclasses are respectively activated by 9 A- and B-ephrin ligands. EphA4 is the only receptor capable of binding to all 9 ephrins and small molecules with overlapped interfaces.

Results: We first determined the structures of the EphA4 ligand binding domain (LBD) in two crystals of P1 space group. Noticeably, 8 EphA4 molecules were found in one asymmetric unit and consequently from two crystals we obtained 16 structures, which show significant conformational variations over the functionally critical A-C, D-E, G-H and J-K loops. The 16 new structures, together with previous 9 ones, can be categorized into two groups: closed and open forms which resemble the uncomplexed and complexed structures of the EphA4 LBD respectively. To assess whether the conformational diversity over the loops primarily results from the intrinsic dynamics, we initiated 30-ns molecular dynamics (MD) simulations for both closed and open forms. The results indicate that the loops do have much higher intrinsic dynamics, which is further unravelled by NMR H/D exchange experiments. During simulations, the open form has the RMS deviations slightly larger than those of the closed one, suggesting the open form may be less stable in the absence of external contacts. Furthermore, no obvious exchange between two forms is observed within 30 ns, implying that they are dynamically separated.

Conclusions: Our study provides the first experimental and computational result revealing that the intrinsic dynamics are most likely underlying the conformational diversity observed for the EphA4 LBD loops mediating the binding affinity and specificity. Interestingly, the open conformation of the EphA4 LBD is slightly unstable in the absence of it natural ligand ephrins, implying that the conformational transition from the closed to open has to be driven by the high-affinity interaction with ephrins because the weak interaction with small molecule was found to be insufficient to trigger the transition. Our results therefore highlight the key role of protein dynamics in Eph-ephrin signalling and would benefit future design of agonists/antagonists targeting Eph receptors.

Figure 1

Crystal structures of the EphA4 LBD. (a) Packing relationship of 8 structures of the EphA4 LBD in one asymmetric unit of P₁ space group. The EphA4 molecule with (red) or without (blue) its high affinity ephrin binding pocket closely contacting the G-H loop of another EphA4 molecule in the same (b) or a neighboring (c) unit.

Figure 2

Comparison of 16 structures. (a) Superimposition of 16 structures of the EphA4 LBD, which can be divided into the closed and open forms based on the conformations of D-E and J-K loops. The color codes for Helix-Sheet-Loop are Red-Yellow-Green for the closed and Cyan-Purple-Brown for the open forms respectively. (b) Superimposition of 11 structures of the EphA4 LBD in the closed form. (c) Superimposition of 5 structures of the EphA4 LBD in the open form.

Figure 3

Comparison with previously-determined structure. (a) Superimposition of 11 present structures (red) with previously-determined 3 structures (blue; 3KH and 2WO1) of the EphA4 LBD in the closed form. (b) Superimposition of 5 present structures (green) with previously-determined 6 structures (3KH, 3GXU, 2WO2, 2WH3 and ref. [15]) of the EphA4 LBD in the open form. The open structures in the uncomplexed state are colored in red while the open structures complexed with ephrin are in blue.

Figure 4

Trajectories of MD simulations. (a-c). Root-mean-square deviations (RMSD) of the heavy atoms for three independent MD simulations for the closed (blue) and open (red) forms. (d-f). Root-mean-square fluctuations (RMSF) of the Cα atoms computed for three independent simulations for the closed (blue) and open (red) forms. The average values and standard deviations over 30-ns simulations are computed and displayed.

Figure 5

Detailed dynamical behaviours. The closed (a and c) and open (b and d) structures of the EphA4 LBD in which green is used for coloring residues with their RMSF values > average value and red for the residues with their RMSF values > 2 average value. (e-g) Structure snapshots (one structure for 3-ns interval) of three independent MD simulations respectively for the closed (blue) and open (brown) forms.

Figure 6

Trajectories of secondary structures of the J-K loop residues. Three independent trajectories of secondary structures of the J-K loop residues (125-143) of the closed (a-c) and open (d-f) forms during 30-ns simulations.

Figure 7

Comparison between crystal structures and simulation ensembles. (a) Superimposition of 14 crystal structures (red) with 10 structure snapshots (blue, one structure for 3-ns interval) in the closed form. (b) Superimposition of 11 crystal structures (green) with 10 structure snapshots (brown, one structure for 3-ns interval) in the open form.

Figure 8

NMR hydrogen-deuterium (H/D) exchange. (a) Superimposition of the ¹H-¹⁵N NMR HSQC spectra of the ¹⁵N-labeled EphA4 LBD at 25°C in the buffer (blue) and 15 min (red) after the lyophilized EphA4 LBD powder was re-dissolved in D₂O. (b) Superimposition of the ¹H-¹⁵N NMR HSQC spectra of the ¹⁵N-labeled EphA4 LBD at 25°C, 15 min (blue) and 2 hr (red) after the lyophilized EphA4 LBD powder was re-dissolved in D₂O. (c) The structure of the EphA4 LBD with the H/D exchange results mapped onto. Blue: the residues completely exchanged within 15 min; green: residues completely exchanged from 15 min to 2 hr; red: residues un-exchanged after 2 hr.