Oligomeric State and Drug Binding of the SARS-CoV‑2 Envelope Protein Are Sensitive to the Ectodomain

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