PIP2 promotes 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 antioncogenic 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 styrene maleic acid lipid particles to measure dimerization in membranes. We observed that the signaling lipid PIP 2 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 TMs are almost parallel, and the positively charged JM segments are expected to be close to each other, causing electrostatic repulsion. The mechanism PIP 2 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 PIP 2 might directly exert a regulatory effect on EphA2 activation in cells that is specific to the ligand-independent conformation of the receptor.

The C. albicans virulence factor Candidalysin polymerizes in solution to form membrane pores and damage epithelial cells

“…OCD can discriminate between helices aligned along the membrane plane and transmembrane (TM) helices (15) (Figure 5C, inset). The OCD data of CL displayed low intensity, and a minimum at ~225 nm and a maximum at ~195 nm, similarly to the expected result for TM helices (16,17). The OCD spectrum therefore indicates that the predominant orientation of helices in CL in membranes was inserted across the membrane, in a TM orientation.…”

Slow conformational dynamics of the human A2A adenosine receptor are temporally ordered

“…Single-molecule fluorescence data were recorded using a customized prism-based TIRF imaging system based on an inverted IX73 microscope (Olympus) and customized TIRF stage (TIRF Labs Inc., Cary, NC, U.S.A.) as described in earlier publications (Berezhna et al, 2012;Stefanski et al, 2021). Quartz microscope slides (G. Finkenbeiner, Waltham MA) and coverslips were cleaned and passivated with polyethylene glycol (m-PEG-SVA) and 3% biotin-PEG-SVA (Laysan Bio Inc, Arab, AL) as previously described (Lamichhane et al, 2010).…”

Biological insights from SMA-extracted proteins

“…EphA2 transmembrane peptide was reconstituted into proteoliposomes with different bilayer thickness, to bias the dimerisation towards one of the two binding modes, and these proteoliposomes were solubilised with SMA for single molecule fluorescence studies. Addition of PIP 2 was found to increase the dimer:monomer ratio only in the thicker bilayer, suggesting that PIP signalling may modulate the pro-oncogenic ligand-independent signalling of EphA2 [ 78 ].…”