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 ¹³C-labeled phospholipids from yeast. We focused on phosphatidylinositol (PI), an important lipid involved in cellular signaling and membrane trafficking. Using ¹³C-labeled PI, we investigated its interaction with the SARS-CoV-2 envelope protein E. ¹³C chemical shifts, T₁ 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 ¹³C-¹³C 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.