Tyrosine phosphorylation and the inhibitory C-terminal SAM domain moderately affect transient interactions in a EphA2 cytoplasmic fragment in solution: A combined experimental and molecular modeling study.

Eph receptors, the largest subfamily of single-pass transmembrane receptor tyrosine kinases, play essential roles in development, including axon guidance and cell positioning, and in adult functions such as synaptogenesis. While their canonical signaling is typically repulsive and ligand-dependent, non-canonical activity promotes cell migration and proliferation in several cancers through ligand-independent mechanisms. Proteases associated with neurodegeneration can cleave Eph receptors, generating near full-length intracellular region (ICR) fragments, whose signaling potential remains unclear. Here, we dissect the mechanistic contributions of the sterile α motif (SAM) domain and tyrosine phosphorylation to the behavior of the EphA2 ICR-one of the best-characterized Eph receptors. The ICR expressed in E. coli retains kinase activity and undergoes phosphorylation at tyrosine residues identified in eukaryotic systems. This phosphorylation reduces domain interactions, as measured by microscale thermophoresis. AlphaFold2Multimer models and available crystal structures of EphA2 dimers provide limited mechanistic insights, likely due to crystal packing effects. Using coarse-grained molecular dynamics simulations (Martini 3.0), we find that SAM-kinase interactions are predominantly transient but show clustering in two kinase-domain regions. NMR spectroscopy with assigned EphA2 SAM domains reveals non-canonical ICR contacts, potentially keeping the canonical SAM interface accessible for partner binding. Introduction of the SHIP2 SAM domain results in canonical EphA2-SHIP2 interactions, along with unexpected contacts with the kinase domain. Together, our results highlight the dynamic, phosphorylation-sensitive nature of EphA2 ICR in solution and suggest a versatile signaling capacity for cleaved intracellular fragments in pathological contexts. Significance: Eph receptors are key signaling molecules in development and disease, yet the behavior of their intracellular region (ICR) following receptor cleavage remains poorly understood. This study reveals that the EphA2 ICR, including the sterile α motif (SAM) and kinase domains, adopt transient interactions between several configurations moderately affected by phosphorylation. Through a combination of biophysical assays, molecular dynamics simulations, and NMR spectroscopy, we identify regions of intra- and inter-domain contacts, including non-canonical interactions with partner SAM domains such as SHIP2. These findings provide critical insight into how cleaved EphA2 fragments remain functionally active in the cytoplasm and suggest a versatile, modular signaling capacity that may be relevant in pathological conditions like cancer and neurodegeneration.