Engineering nanomolar peptide ligands that differentially modulate EphA2 receptor signaling

Abstract

Edited by Wolfgang Peti The EPH receptor A2 (EphA2) tyrosine kinase plays an important role in a plethora of biological and disease processes, ranging from angiogenesis and cancer to inflammation and parasitic infections. EphA2 is therefore considered an important drug target. Two short peptides previously identified by phage display, named YSA and SWL, are widely used as EphA2-targeting agents owing to their high specificity for this receptor. However, these peptides have only modest (micromolar) potency. Lack of structural information on the binding interactions of YSA and SWL with the extracellular EphA2 ligand-binding domain (LBD) has for many years precluded structure-guided improvements. We now report the high-resolution (1.53-2.20 Å) crystal structures of the YSA peptide and several of its improved derivatives in complex with the EphA2 LBD, disclosing that YSA targets the ephrin-binding pocket of EphA2 and mimics binding features of the ephrin-A ligands. The structural information obtained enabled iterative peptide modifications conferring low nanomolar potency. Furthermore, contacts observed in the crystal structures shed light on how C-terminal features can convert YSA derivatives from antagonists to agonists that likely bivalently interact with two EphA2 molecules to promote receptor oligomerization, autophosphorylation, and downstream signaling. Consistent with this model, quantitative FRET measurements in live cells revealed that the peptide agonists promote the formation of EphA2 oligomeric assemblies. Our findings now enable rational strategies to differentially modify EphA2 signaling toward desired outcomes by using appropriately engineered peptides. Such peptides could be used as research tools to interrogate EphA2 function and to develop pharmacological leads.

Allosteric Regulation of the EphA2 Receptor Intracellular Region by Serine/Threonine Kinases

“…We therefore used the fully quantified spectral imaging Forster Resonance Energy Transfer (FSI-FRET) method 41 to examine how EphA2 mutations mimicking or preventing phosphorylation of kinase-SAM linker residues affect EphA2 lateral interactions in the absence of ligand binding in HEK293T cells. We studied eight different mutants, including 5E, 3E, S897E, S892E, 5A (S892A/S897A/T898A/S899A/S901A), ASAAA (S892A/T898A/S899A/S901A), S897A and S892A, and compared them to EphA2 WT 94 . Homodimerization experiments were performed in cells with EphA2 labeled at the C-terminus with fluorescent proteins (either mTurquoise or EYFP, a FRET pair) attached via a flexible linker (GGS)5.…”

Ligand bias in receptor tyrosine kinase signaling

“…The two peptides, designated here YSK (bAWLAYPDSVPYSK-biotin) and YSPK (bAWLAYPDSVPYSPK-biotin), differ only by one residue (P13 in YSPK, which is not present in YSK). Both peptides induce two responses that are also induced by the natural ephrinA ligands (50). Response A involves phosphorylation of tyrosine 588 (Y588) in the juxtamembrane region of EphA2, which is an autophosphorylation site known to promote receptor kinase activity.…”

“…In a previous section, we used equation 1to fit experimental dose-response curves obtained for the EphA2 RTK activated by two engineered peptide ligands and equation 2to calculate bias coefficients and draw conclusions about the existence of bias. EphA2 is activated by the peptide ligands through a different mechanism than VEGFR2 by its ligand VEGFA, since each biotinylated peptide bivalently interacts with two EphA2 molecules to form a complex of two peptides bound to two receptors (50). To investigate whether equations (1) and 2are applicable for analysis of bias in RTKs with different dimerization mechanisms, we first simulated dose-response curves for the cases of bias and no bias for two VEGFR2 ligands ( Figure 3F), and then asked if these two cases can be correctly identified.…”

Approaches to Manipulate Ephrin-A:EphA Forward Signaling Pathway

“…An advantage of peptides is their easy chemical modification that leads to optimized affinity or enhanced agonist potency. For instance, βA-WLA-YRPK-bio, a YSA/SWL modified peptide that is biotinylated at its C-terminal end and contains an additional β-alanine in N-terminus, exhibits both a higher affinity and a higher ability to induce EphA2 signaling due to its strengthened EphA clustering effect [115]. Since the size of EphA clusters controls the strength of the downstream pathway, DNA trimeric nanostructures have been used to couple three non-modified SWL peptides [116].…”

“…Finally, chemical peptide modifications enable the development of antagonist peptidic chains from agonist ones by changing their local charges. Removing the C-terminal negative charge of the YSA agonist derivative βA-WLA-YRPK enhances its affinity and suppress its ability to activate EphA2 signaling, turning it into a competitive antagonist [115].…”