Pentameric Architecture of the SARS-CoV-2 Envelope Protein Revealed by SEC-MALS, Cryo-EM, and Molecular Dynamics.

The SARS-CoV-2 envelope (E) protein is a small viroporin that drives viral assembly, budding, and host interactions, yet its structural organization has remained elusive. Earlier nuclear magnetic resonance spectroscopy studies hint at oligomerization without direct evidence, and the construct lacks the flexible C-terminal region. To bridge this gap, we synthesized the full-length E protein to investigate its oligomeric state. Using size-exclusion chromatography coupled with multiangle light scattering, we demonstrated that the E protein assembles as a stable pentamer in solution. We then reconstituted the E protein into membrane scaffold protein nanodiscs to mimic the lipid bilayer environment for structural analyses by negative-stain electron microscopy and cryo-electron microscopy, which revealed pentamer-like features. Molecular dynamics simulations of the E protein in a nanodisc and a membrane bilayer setting further corroborated the structural flexibility of the C-terminal domain. Collectively, these data present direct evidence that the SARS-CoV-2 E protein assembles as a pentamer in both solution and membrane-mimetic environments. Our results provide a structural foundation for future investigations into the E protein's roles in ion channel activity, membrane remodeling, and virus-host interactions.