Conformational clamping by a membrane ligand activates the EphA2 receptor

Abstract

The EphA2 receptor is a promising drug target for cancer treatment, since EphA2 activation can inhibit metastasis and tumor progression. It has been recently described that the TYPE7 peptide activates EphA2 using a novel mechanism that involves binding to the single transmembrane domain of the receptor. TYPE7 is a conditional transmembrane (TM) ligand, which only inserts into membranes at neutral pH in the presence of the TM region of EphA2. However, how membrane interactions can activate EphA2 is not known. We systematically altered the sequence of TYPE7 to identify the binding motif used to activate EphA2. With the resulting six peptides, we performed biophysical and cell migration assays that identified a new potent peptide variant. We also performed a mutational screen that determined the helical interface that mediates dimerization of the TM domain of EphA2 in cells. These results, together with molecular dynamic simulations, allowed to elucidate the molecular mechanism that TYPE7 uses to activate EphA2, where the membrane peptide acts as a molecular clamp that wraps around the TM dimer of the receptor. We propose that this binding mode stabilizes the active conformation of EphA2. Our data, additionally, provide clues into the properties that TM ligands need to have in order to achieve activation of membrane receptors.

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

“…Even differences in pH can be mimicked by defining different charged states for acidic/basic groups, or by using the Titratable Martini approach (Grünewald et al, 2020). Moreover, trimers or oligomer can be accurately studied (Westerfield et al, 2021) with Martini 3 or even interactions with larger protein complexes (Liaci et al, 2021). Additional simulations and experimental studies are needed to delineate the possible effects of such variables on the TM dimer structural configurations and their stability.…”

Allosteric inhibition of the epidermal growth factor receptor through disruption of transmembrane interactions

“…The transition between states occurred in a sigmoidal fashion with a midpoint (pH50) of 5.00 ± 0.01 (24). For pH-responsive peptides that bind the TM of an RTK, the presence of that RTK can increase the pH50 due to an increase in tendency to be inserted in the presence of a membrane binding partner (15,23). For this reason, we repeated the experiment using proteo-liposomes containing a peptide mimic of the TM of EGFR (TM-EGFR).…”

“…Alves et al (15) evolved this concept and designed the peptide TYPE7 to be specific for the RTK EphA2. TYPE7 binds the TM to allosterically regulate EphA2 kinase activity by causing a configurational change (23). This method of regulation is likely to be more selective for EphA2 than targeting the highly conserved kinase domain.…”

Structural and Functional Insights into the Transmembrane Domain Association of Eph Receptors

“…Therefore, understanding the interactions involved in TM-TM recognition inside the membrane is an important challenge. Moreover, recent studies have demonstrated how the activity of specific Ephs may be controlled by small peptides that specifically recognize their TMDs, and thereby change how the TM helices bind to one another (their configuration) [27,28]. Thus, the conserved role of TMDs in regulating the function of Eph receptors makes them promising targets for therapeutic intervention.…”

“…Similarly, the recently designed TYPE7 peptide is also highly soluble in the aqueous solution that inserts into cellular membranes at slightly acidic pH. The TM state of TYPE7 interacts with EphA2 and induces receptor oligomerization and phosphorylation [27,28]. Using CG MD simulation our laboratory has helped to suggest a mechanism by which the TYPE7 peptide stabilizes the active configuration of the helix dimer by forming a 2:1 (EphA2 TMs: peptide) trimer complex and thereby promoting EphA2 oligomerization.…”