The Cytoplasmic Domain of the SARS- CoV-2 Envelope Protein Assembles into a b-Sheet Bundle in Lipid Bilayers

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pen-tameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a j3-sheet -rich conformation that contains three j3-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water -edited NMR spectra indicate that the third j3-strand at the C terminus of the protein is well hydrated, indi-cating that it is at the surface of the j3-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing inter-molecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt j3-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the j3-strand conformation and the previously reported a-helical conformation may underlie the multiple functions of E in the host cell and in the virion.(c) 2023 Elsevier Ltd. All rights reserved.