Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix

Abstract

Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.

Protein Amphipathic Helix Insertion: A Mechanism to Induce Membrane Fission

“…Scission was not observed on GUVs containing PtdIns(4,5)P 2 (Gopaldass et al, 2017) Normal M2 from influenza virus Cholesterol M2 binds cholesterol specifically (Ekanayake et al, 2016;Elkins et al, 2017Elkins et al, , 2018 Cholesterol significantly augmented the capability of the M2 amphipathic helix (M2AH) in inducing bilayer pits. POPG does not have the ability of significantly impacting M2AH-induced membrane modulation (Pan et al, 2019) Reverse…”

“…(3) PtdIns3P and PtdIns(3,5)P 2 for Atg18 (Gopaldass et al, 2017;Scacioc et al, 2017); (4) cholesterol for M2 protein from influenza A virus (Ekanayake et al, 2016;Elkins et al, 2017Elkins et al, , 2018Herneisen et al, 2017;Pan et al, 2019; and references therein), see Table 2.…”

“…Reverse-topology membrane fission-inducing protein M2 from influenza A virus contains an AH, but uses neutral cholesterol as a cofactor in the membrane fission process (Elkins et al, 2017;Herneisen et al, 2017;Pan et al, 2019; and references therein), see Table 2. However, we should keep in mind that in the case of reverse topology fission, AHs insert into the proximal leaflet of the budding membrane-enclosed compartment, and not into the distal leaflet, as in the case of the normal-topology fission.…”

Phosphatidic acid in membrane rearrangements

“…Such lipid‐mediated regulations of membrane rearrangements are only beginning to be explored. Sterols ( and references therein), phosphoinositides ( and references therein), PA and DAG ( and references therein) are among few lipids that have regulatory role in membrane fusion and fission.…”

Influenza A M2 Spans the Membrane Bilayer, Perturbs its Organization and Differentiates the Effect of Amantadine and Spiro[pyrrolidine-2,2'-adamantane] AK13 on Lipids

“…These effects induce changes in the mobility and packing of the lipids and the curvature of the lipid bilayers. 19,[27][28] In a previous work, the effects of the M2 WT channel blockers Amt and AK13 on DMPC bilayers ( Figure 1) were compared using DSC, x-ray scattering, ssNMR spectroscopy, and MD simulations. 29 It was found that the two drugs localized in the polar head area of the DMPC bilayers, stabilized through hydrogen bonding with sn-2 carbonyls in their amine form, and phosphate oxygens in their ammonium form.…”