Mapping of the binding surface between EPHA5 and antagonist peptide by NMR spectroscopy

Abstract

The Eph receptors constitute the largest family of receptor tyrosine kinases, with 16 individual receptors that are activated by 9 different ephrins throughout the animal kingdom. Eph receptors and their ligands are both anchored to the plasma membrane, and are subdivided into two subclasses (A and B) based on their sequence conservation and binding preferences. The critical roles of Eph-ephrin mediated signalling in various physiological and pathological processes mean that the interface at which the interaction between receptor and ligand occurs is a promising target for the development of molecules to treat human diseases, such as neuron regeneration, bone remodelling diseases, and cancer. A diverse spectrum of peptides that act as antagonists of Eph-ephrin with differential selectivity has previously been identified. One of these peptides, called WDC, is attractive because it has been found to antagonize the interaction between EphA5 and its ligands with high selectivity. EphA5 receptor and its ligands serve as repulsive axonguidance cues in the developing brain. Their interaction triggers growth cone collapse and inhibits the neurite outgrowth in vitro. Furthermore, abnormal expression of these molecules would result in the disruption of axonal path finding and mid-line crossing in vivo. So far, the three-dimensional structure of the EphA5 ligand-binding domain has not been determined. In the present study, the crystal of EphA5 ligand-binding domain was obtained. Structural characterizations of both EphA5 and WDC were assessed by CD and NMR. Furthermore, characterizations of binding interactions between EphA5 and WDC peptide were characterized by NMR and ITC. The binding surface between EphA5 and WDC was demonstrated using NMR. Interestingly, WDC was found to be well-folded even in the free-state. Its binding surface for EphA5 receptor was mapped by Ala site-directed mutagenesis and NMR titration. Taken all together, our results may provide critical rationales for further design of specific EphA5 antagonists for various therapeutic applications.