Abstract
The 16 EphA and EphB receptors represent the largest family of receptor tyrosine kinases, and their interactions with 9 ephrin-A and ephrin-B ligands initiate bidirectional signals controlling many physiological and pathological processes. Most interactions occur between receptor and ephrins of the same class, and only EphA4 can bind all A and B ephrins. To understand the structural and dynamic principles that enable Eph receptors to utilize the same jellyroll β-sandwich fold to bind ephrins, the VAPB-MSP domain, peptides and small molecules, we have used crystallography, NMR and molecular dynamics (MD) simulations to determine the first structure and dynamics of the EphA5 ligand-binding domain (LBD), which only binds ephrin-A ligands. Unexpectedly, despite being unbound, the high affinity ephrin-binding pocket of EphA5 resembles that of other Eph receptors bound to ephrins, with a helical conformation over the J–K loop and an open pocket. The openness of the pocket is further supported by NMR hydrogen/deuterium exchange data and MD simulations. Additionally, the EphA5 LBD undergoes significant picosecond-nanosecond conformational exchanges over the loops, as revealed by NMR and MD simulations, but lacks global conformational exchanges on the microsecond-millisecond time scale. This is markedly different from the EphA4 LBD, which shares 74% sequence identity and 87% homology. Consequently, the unbound EphA5 LBD appears to comprise an ensemble of open conformations that have only small variations over the loops and appear ready to bind ephrin-A ligands. These findings show how two proteins with high sequence homology and structural similarity are still able to achieve distinctive binding specificities through different dynamics, which may represent a general mechanism whereby the same protein fold can serve for different functions. Our findings also suggest that a promising strategy to design agonists/antagonists with high affinity and selectivity might be to target specific dynamic states of the Eph receptor LBDs.
Highlights
The Eph receptors constitute the largest family of receptor tyrosine kinases, with 16 members in vertebrates, which can be activated by 9 ephrin ligands [1,2,3,4,5,6]
The PCR fragment was cloned into a modified pET32a vector (Novagen), and subsequently the vector was transformed into E. coli Rosetta-gami (DE3) cells (Novagen) as we previously performed for the EphA4 ligand-binding domain (LBD) [14,17], which allows more efficient formation of disulfide bonds and expression of eukaryotic proteins containing codons rarely used in E. coli
Hydrogen-deuterium (H/diffusion coefficients (Ds)) exchange experiments were conducted on the EphA5 LBD to gain an initial insight into its dynamic behavior on the ms-hr time scale, as we previously described for human ephrin-B2 and EphA4 [12]
Summary
The Eph receptors constitute the largest family of receptor tyrosine kinases, with 16 members in vertebrates, which can be activated by 9 ephrin ligands [1,2,3,4,5,6]. Eph receptors and ephrins are both anchored onto the plasma membrane, and the interactions between them initiate bidirectional signals that direct pattern formation and morphogenetic processes such as axon growth, cell assembly and migration, and angiogenesis [1,2,3,4,5,6,7,8]. As they function in both physiology and disease, Eph receptors and ephrins represent promising targets for drug design. EphA4 is the only receptor capable of interacting with all 9 ephrins of both A and B classes [7]
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