Determining Cholesterol Binding to Membrane Proteins by Cholesterol ¹³C Labeling in Yeast and Dynamic Nuclear...

We present a general strategy for determining the cholesterol-binding site of eukaryotic membrane proteins in native-like lipid membranes by NMR spectroscopy. The strategy combines yeast biosynthetic ¹³C enrichment of cholesterol with detection of protein-cholesterol ¹³C-¹³C cross peaks in 2D correlation NMR spectra under the dynamic nuclear polarization (DNP) condition. Low-temperature DNP not only allows high-sensitivity detection of weak protein-cholesterol cross peaks in 2D spectra but also immobilizes cholesterol and protein to enable intermolecular distance measurements. We demonstrate this approach on the influenza M2 protein, which utilizes cholesterol to conduct membrane scission in the last step of virus budding and release from the host cell plasma membrane. A ¹³C-¹³C double-quantum filter was employed to significantly simplify the 2D ¹³C-¹³C correlation spectra and facilitate the identification of protein-cholesterol cross peaks. A number of cross peaks between the M2 transmembrane residues' side chains and the cholesterol sterol group were detected, which complement recently measured protein contacts to the isooctyl tail of cholesterol to define an extended binding interface. These results provide atomic-level evidence of M2-cholesterol interaction to cause membrane curvature and scission, and the approach is generally applicable to other eukaryotic membrane proteins for understanding the influence of cholesterol on membrane protein function.