The influenza M2 protein forms a drug-targeted tetrameric proton channel to mediate virus uncoating and carries out membrane scission to mediate virus release. While the proton channel function of M2 has been extensively studied, the mechanism by which M2 conducts membrane scission is still not well understood. Previous fluorescence and electron microscopy studies indicated that M2 tetramers concentrate at the neck of the budding virus in the host plasma membrane. However, molecular evidence for this clustering is scarce. Here, we use F solid-state NMR to investigate M2 clustering in phospholipid bilayers. By mixing equimolar amounts of 4F-Phe47 labeled M2 and CF-Phe47 labeled M2 and measuring F-CF cross peaks in 2D FF correlation spectra, we show that M2 tetramers form nanometer-scale cluster in lipid bilayers. This clustering is stronger in cholesterol-containing membranes and phosphatidylethanolamine (PE) membranes than in cholesterol-free phosphatidylcholine and phosphatidylglycerol membranes. The observed correlation peaks indicate that Phe47 sidechains from different tetramers are less than ~2 nm apart. HF correlation peaks between lipid chain protons and fluorinated Phe47 indicates that Phe47 is more deeply inserted into the lipid bilayer in the presence of cholesterol than in its absence, suggesting that Phe47 preferentially interacts with cholesterol. Static P NMR spectra indicate that M2 induces negative Gaussian curvature in the PE membrane. These results suggest that M2 tetramers cluster at cholesterol- and PE-rich regions of cell membranes to cause membrane curvature, which in turn can facilitate membrane scission in the last step of virus budding and release.Copyright © 2021. Published by Elsevier B.V.
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