Phosphatidylinositol Interactions with the SARS-CoV-2 Envelope Protein Investigated by Lipid 13 C Labeling and Solid-State NMR

Membrane protein structure and function are intimately influenced by the surrounding lipids. Solid-state NMR spectroscopy is an important approach for investigating site-specific protein–lipid interactions under physiological conditions. To observe protein–lipid contacts with high spectral sensitivity and lipid selectivity, here we describe an efficient protocol for producing and purifying 13 C-labeled phospholipids from yeast. We focused on phosphatidylinositol (PI), an important lipid involved in cellular signaling and membrane trafficking. Using 13 C-labeled PI, we investigated its interaction with the SARS-CoV-2 envelope protein E. 13 C chemical shifts, T 1 relaxation times and cross-polarization buildup times indicate that the E transmembrane domain (ETM) rigidified the inositol headgroup and the acyl chains without causing significant chemical shift perturbations to the lipid, indicating that ETM–PI interaction is weak. Despite this weak interaction, protein–lipid cross peaks are observed in two-dimensional 13 C– 13 C correlation spectra, indicating that a subpopulation of PI lipids has specific interactions with the protein. These protein-bound annular PI lipids interact with Thr, Asn and Ser residues at the N- and C-terminal ends of the transmembrane helix, likely through hydrogen bonding and other electrostatic interactions. These results provide direct evidence that the SARS E protein interact with anionic PI lipids, and this interaction may modulate cation conduction by this pathogenic viroporin.