Oligomeric State and\nDrug Binding of the SARS-CoV‑2\nEnvelope Protein Are Sensitive to the Ectodomain

The envelope (E) protein of SARS-CoV-2 is the smallest\nof the three\nstructural membrane proteins of the virus. E mediates budding of the\nprogeny virus in the endoplasmic reticulum Golgi intermediate compartment\nof the cell. It also conducts ions, and this channel activity is associated\nwith the pathogenicity of SARS-CoV-2. The structural basis for these\nfunctions is still poorly understood. Biochemical studies of E in\ndetergent micelles found a variety of oligomeric states, but recent <sup>19</sup>F solid-state NMR data indicated that the transmembrane domain\n(ETM, residues 8–38) forms pentamers in lipid bilayers. Hexamethylene\namiloride (HMA), an E inhibitor, binds the pentameric ETM at the lipid-exposed\nhelix–helix interface. Here, we investigate the oligomeric\nstructure and drug interaction of an ectodomain-containing E construct,\nENTM (residues 1–41). Unexpectedly, <sup>19</sup>F spin diffusion\nNMR data reveal that ENTM adopts an average oligomeric state of dimers\ninstead of pentamers in lipid bilayers. A new amiloride inhibitor,\nAV-352, shows stronger inhibitory activity than HMA in virus-like\nparticle assays. Distance measurements between <sup>13</sup>C-labeled\nprotein and a trifluoromethyl group of AV-352 indicate that the drug\nbinds ENTM with a higher stoichiometry than ETM. We measured protein–drug\ncontacts using a sensitivity-enhanced two-dimensional <sup>13</sup>C–<sup>19</sup>F distance NMR technique. The results indicate\nthat AV-352 binds the C-terminal half of the TM domain, similar to\nthe binding region of HMA. These data provide evidence for the existence\nof multiple oligomeric states of E in lipid bilayers, which may carry\nout distinct functions and may be differentially targeted by antiviral\ndrugs.