Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR

Cholesterol is important for membrane protein function, but cholesterol-binding structures of membrane proteins are difficult to determine by X-ray crystallography and electron microscopy due to the small size and dynamic nature of cholesterol. We have developed a solid-state NMR approach to determine the cholesterol-binding structure of membrane proteins in lipid bilayers. Applied to the influenza M2 protein, the measured interatomic distances and cholesterol orientational angles indicate that cholesterol binds M2 in a substoichiometric fashion, flanking methyl-rich transmembrane (TM) residues near an amphipathic helix, without requiring a cholesterol recognition sequence motif, and this substoichiometric binding uniquely correlates with membrane curvature generation. These results give unprecedented insights into how cholesterol clusters M2 to the neck of the budding virus to mediate membrane scission.