Molecular Determinants of Receptor-Specific Membrane Interactions in EphA1 and EphA2 from Coarse-Grained Simulations

Abstract Ephrin receptors (Ephs) are receptor tyrosine kinases that regulate cellular growth, differentiation, and motility. EphA2, often overexpressed in cancer, is notable for its ligand-independent activation, which drives pro-oncogenic signaling distinct from the canonical, ligand-dependent pathway that restricts cell movement. While ligand binding induces extracellular clustering, kinase activation depends on dimerization within the transmembrane (TM) region. EphA1 and EphA2 differ substantially in their function, likely due to differences in their TM, but more so in their juxtamembrane (JM), and membrane-proximal fibronectin type III (FN1/FN2) domains. How these latter two regions modulate dimerization has not yet been investigated. To address this, we performed extensive coarse-grained (CG) simulations using Martini3 in an anionic POPC/PS/PIP2 model of the plasma membrane. Both receptors formed stable TM dimers, though EphA1 favored a symmetric AXXXGXXXG-centered interface, whereas EphA2 favored an alternate leucine zipper interface. All protein constructs sampled multiple configurations, reflecting substantial intrinsic variability. AlphaFold3 does not yield reliable predictions and cannot account for the effects of the lipid bilayer. In the CG simulations, basic residues in the JM region remained membrane-bound, and the EphA2 FN domain displayed sustained PIP2 interactions, consistent with previous observations. Notably, constructs with the FN2 domain alone (a fragment relevant to Alzheimer's Disease) restricted TM association in both receptors, whereas inclusion of the second FN1 domain restored dimerization but produced receptor-specific extracellular interfaces. These differences arise from distinct FN1-FN2 linker flexibilities, a different level of sequence conservation of EphA1 compared to several other Ephs and FN-domain membrane contacts, which together shape TM geometry and lipid engagement. Our results predict how TM and TM-proximal elements cooperatively tune dimerization in Eph receptors. This work offers a molecular rationale for the divergent activation behaviors of EphA1 and EphA2 and provides testable models relevant to cancer and neurodegeneration biology as well as Eph-driven signaling.