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

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric 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 ß-sheet-rich conformation that contains three ß-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 ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular 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 ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

Deducing the Crystal Structure of MERS-CoV Helicase.

RNA virus encodes a helicase essential for viral RNA transcription and replication when the genome size is larger than 7 kb. Coronavirus (CoV) has an exceptionally large RNA genome (~30 kb) and it encodes an essential replicase, the nonstructural protein 13 (nsp13), a member of superfamily 1 helicases. Nsp13 is among the evolutionary most conserved proteins not only in CoVs but also in nidovirales. Thus, it is considered as an important drug target. However, the high-resolution structure of CoV nsp13 remained unavailable even until more than a decade after the outbreak of the severe acute respiratory syndrome coronavirus (SARS-CoV) in 2003, which hindered the structure-based drug design. This is in part due to the intrinsic flexibility of nsp13. Here, we describe protocols of deducing the crystal structure of Middle East respiratory syndrome coronavirus (MERS-CoV) helicase in detail, which include protein expression, purification, crystallization, enzymatic characterization, and structure determination. With these methods, catalytically active recombinant MERS-CoV nsp13 protein can be prepared and crystallized and the crystal structure can be solved.