Conserved Transmembrane Asparagine Is Essential for the Ion-Conducting Structure and Dynamics of the SARS-CoV-2...

The envelope (E) protein of the SARS coronavirus forms a pathogenic cation-selective channel across intracellular membranes. The ion-conduction mechanism of the hydrophobic transmembrane domain of the protein, ETM, has been elusive despite recent determination of its high-resolution structures in the closed and open states. Here, we investigate the structural mechanism of SARS ETM by mutating two residues, T11 and N15. T11A is the second most common mutation in Omicron variants and has attenuated channel activity and cell lethality compared to wild-type ETM, whereas the N15A mutant is absent from SARS-CoV-2 variants and is non-conducting. Using solid-state NMR spectroscopy, we measured the conformation, dynamics, water accessibility, and membrane insertion of these two mutants in cholesterol-containing lipid bilayers that mimic the cell membrane in which E is localized during virus assembly. We found that the T11A mutation caused minimal perturbation to the protein structure but decreased its thermostability. In contrast, the N15A mutation caused large-scale conformational changes to the protein, rigidified the N-terminal segment, and mobilized and disordered the C-terminal segment. These data indicate that this asparagine, conserved across E proteins of many coronaviruses, is essential for the assembly of the transmembrane helical bundle and for the conformational dynamics of the protein. Together with previous findings about wild-type ETM, we propose a transporter model for the SARS E protein where the N- and C-terminal polar segments couple allosterically through the conserved asparagine to achieve the proper helical packing and conformational dynamics for cation conduction.