PIP₂promotes conformation-specific dimerization of the EphA2 membrane region

Abstract

The impact of the EphA2 receptor on cancer malignancy hinges on the two different ways it can be activated. EphA2 induces anti-oncogenic signaling after ligand binding, but ligand-independent activation of EphA2 is pro-oncogenic. It is believed that the transmembrane (TM) domain of EphA2 adopts two alternate conformations in the ligand-dependent and the ligand-independent states. However, it is poorly understood how the difference in TM helical crossing angles found in the two conformations impacts the activity and regulation of EphA2. We devised a method that uses hydrophobic matching to stabilize two conformations of a peptide comprising the EphA2 TM domain and a portion of the intracellular juxtamembrane (JM) segment. The two conformations exhibit different TM crossing angles, resembling the ligand-dependent and ligand-independent states. We developed a single-molecule technique using SMALPs to measure dimerization in membranes. We observed that the signaling lipid PIP2 promotes TM dimerization, but only in the small crossing angle state, which we propose corresponds to the ligand-independent conformation. In this state the two TM are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP2 uses to promote dimerization might involve alleviating this repulsion due to its high density of negative charges. Our data reveal a conformational coupling between the TM and JM regions, and suggest that PIP2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

Transmembrane region dimer structures of Type 1 receptors readily sample alternate configurations: MD simulations using the Martini 3 coarse grained model compared to AlphaFold2 Multimer

“…For example, it is known that negatively charged lipids, such as POPS and cell signalling lipids PIP2 and PIP3 alter the functional behavior of TM-but also TM peripheral proteins cells (Abd Halim et al, 2015;Cao et al, 2019;Yen et al, 2018). Specifically, it is noticeable that almost all of the 11 systems chosen here contain a cationic "plug" to prevent sliding of the C-terminal region into the membrane and recent work by Barerra and colleagues (Stefanski et al, 2021) suggests that EphA2 may switch from a parallel TM helix dimer in a wider membrane with PIP2, where positive charges can be tolerated to the structure with a wider crossing angle in a thinner membrane without PIP2, where the juxtamembrane regions may repulse. In this sense the NMR studies of and the predictions/simulations of TM dimers here seem rather artificial since only neutral zwitterionic detergents/lipids were used.…”

Conformational clamping by a membrane ligand activates the EphA2 receptor

“…Interface 1 corresponds to the helix-helix contacts observed in the NMR structure of the TM EphA2 dimer [9]. This interface is mediated by a heptad repeat (HR) motif [4] and is proposed to be populated when EphA2 undergoes ligand-independent (non-canonical) activation [10]. Interface 2 is separated by a ~140 degree rotation around the helical axis from interface 1.…”