The erythropoietin-producing hepatocellular carcinoma (Eph) receptors constitute the largest family of receptor tyrosine kinases, with 16 members throughout the animal kingdom, which are activated by 9 ephrin ligands [1
]. Eph receptors and their ephrin ligands are both anchored onto the plasma membrane, which are subdivided into two subclasses, (A and B), based on their sequence conservation and binding preferences. In general, EphA receptors (EphA1-A10) only interact with glycosylphosphatidylinositol (GPI)-anchored ephrin-A ligands (ephrin-A1-A6), while EphB receptors (EphB1-B6) interact with transmembrane ephrin-B ligands (ephrin-B1-ephrin-B3) that have a short cytoplasmic domain carrying both SH2 and PDZ domain-binding motifs [7
]. Interactions between Eph receptors and ephrins initiate bidirectional signals which direct pattern formation and morphogenetic processes, such as axon growth, cell assembly and migration, and angiogenesis [1
]. The roles of Eph receptors and ephrins in bone remodelling, immune function, and blood clotting, and stem cells, are also starting to be characterized.
All Eph receptors share the same modular structure, consisting of a unique N-terminal ephrin binding domain followed by a cysteine-rich linker and two fibronectin type III repeats in the extracellular region. The intracellular region is composed of a conserved tyrosine kinase domain, a C-terminal sterile α-domain, and a PDZ binding motif. The N-terminal 180-residue globular domain of the Eph receptors has been shown to be sufficient for high-affinity ephrin binding [9
], thus called the ligand binding domain (LBD). So far, structures have been determined for the Eph LBD in the free state [9
], in the complexed forms between A-receptors and A-ephrins [12
]; A-receptors and B-ephrins [13
]; B-receptors and B-ephrins [11
] and B-receptors and A-ephrins [20
], as well as between receptors and antagonistic peptides [21
]. The ligand binding domains of both EphA and EphB receptors adopt the same jellyroll β-sandwich architecture composed of 11 antiparallel β-strands connected by loops of various lengths. On the other hand, the ectodomain of the ephrins is also conserved and consists of an eight-stranded β-barrel with a Greek key topology, including several large and highly conserved functional loops, such as the G-H and C-D loops [11
], which are highly dynamic in solution as revealed by a NMR study [23
]. The common structural feature observed in Eph-ephrin complexes is the insertion of the solvent-exposed and dynamic ephrin G-H loop into the Eph receptor hydrophobic channel formed by the convex sheet of four β-strands capped by the D-E, J-K, and G-H loops. Nevertheless, additional interactions such as the involvement of the A-C loop fine-tune the affinity and specificity of the binding cross subclasses [18
Interactions between the Eph receptors and ephrins of the same subclass are quite promiscuous but interactions between subclasses are relatively rare. EphA4 is the only receptor capable of interacting with all 9 ephrins of both A- and B-subclasses to mediate a diverse spectrum of biological activities [24
]. While EphA4 interacts with ephrin-A ligands to mediate a variety of critical biological processes, such as inhibiting integrin downstream signaling pathways and modulating sensory and motor projections [25
], it is also able to bind all three ephrin-B ligands. For example, EphA4 interacts with ephrin-B1 expressed in human platelets to stabilize blood clot formation through an integrin-dependent mechanism [28
]. By interacting with ephrin-B2 and/or ephrin-B3, EphA4 regulates neuronal circuits important for coordinated movement and may inhibit the regeneration of injured spinal cord axons [29
]. As a consequence, EphA4 was also considered as a promising target for the development of small molecule drugs to treat human diseases [14
The unique ability for the EphA4 LBD to bind all 9 ephrins with similar interfaces renders it to be an attractive model for deciphering the fundamental principle governing protein-ligand interactions. Currently, our understanding of molecular recognition is still incomplete, and in particular the role of protein dynamics in mediating binding affinity and specificity remains to be delineated. Previously, 9 crystal structures were determined for the EphA4 LBD in the free state [13
] and in complex with ephrinA2 and ephrinB2 [13
]. The most outstanding observation is that while the jellyroll β-sandwich core is highly similar in all these structures, the loops, especially D-E, G-H and J-K loops critical for binding, have dramatic conformational variations, which is largely unexpected for such a small protein [15
]. This implies that the functionally critical loops might have higher dynamics but it remains to be clarified that the variations of loop conformations are not primarily due to the crystal packing force or/and differential crystallization conditions.
In the present study, we obtained two crystals of the EphA4 LBD at P1 space group and subsequently determined their structures. Remarkably, 8 EphA4 chains were found in one asymmetric unit and as a result we gained 16 new structures from the two crystals. Although the 16 structures have an almost identical conformation over the jellyroll β-sandwich core, they display significant variations over the A-C, D-E, G-H and J-K loops, which consequently led to the classification of the 16 structures into two groups: closed and open forms based on the conformations of the D-E and J-K loops. To gain insight into the dynamical behaviours and the relationship between the closed and open forms, we initiated 30-ns molecular dynamics (MD) simulations for two structures, which represents the closed and open forms respectively. The obtained results show that indeed the loops do have much larger intrinsic dynamics than the rest of the molecules, which was further supported by NMR hydrogen-deuterium (H/D) exchange experiments.