1. Introduction:
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the
respiratory illness coronavirus disease 19 (COVID-19), which escalated
to a global pandemic in 20201. SARS-CoV-2 is a
positive-sense single-stranded RNA virus made up of four structural
proteins, which include nucleocapsid (N), envelope (E), membrane (M),
and spike (S) glycoproteins2. S glycoprotein,
decorated on the surface of the virus, mediates the virus entry process
by binding to different host cell receptors, which includes the human
angiotensin-converting enzyme 2 (hACE2), making it an important target
for developing vaccines and therapeutics3,4.
S glycoprotein on the mature virion is a homotrimer, with each monomer
comprising two subunits: the S1 subunit containing the N-terminal domain
(NTD) and the receptor-binding domain (RBD) that binds to the hACE2
receptor, and the S2 subunit containing the fusion peptide that mediates
the membrane fusion of the virus and host cells3,5.
Rapid advances in the structural biology of the SARS-CoV-2 S
glycoprotein have occurred since its initial outbreak, including pre-
and post-fusion S glycoprotein, RBD-ACE2 complex, and trimeric
spike-ACE2 complex5-14.
While Rhinolophus affinisrepresent the natural reservoir
of precursors to SARS-CoV-215, multiple species, in
addition to humans, are susceptible to infection16-28.
In April 2020, outbreaks of SARS-CoV-2 on American mink (Mustela
vison ) farms were first reported in the Netherlands, with later
outbreaks observed in mink farms throughout Europe and North
America25,26,29,30. The virus was transmitted to mink
by infected human workers and was capable of transmission among American
mink and back to humans31. In Denmark, sequence
analysis of human samples identified five related clusters with
different mutations in the S glycoprotein, and one of these, cluster 5,
raised alarms internationally for harboring five mutations (∆69-70,
Y453F, D614G, I692V, M1229I) in the S
glycoprotein31,32. A notable mutation present in this
and other clusters is Y453F, located in the RBD of the S glycoprotein,
which arose via parallel evolution in independent mink outbreaks and has
been shown to increase the binding and entry efficiency into cells
expressing American mink ACE2 (mvACE2) while retaining its affinity to
hACE28,32.
Previous studies reported the crystal and cryo-EM structures of mink
variant spike RBDs in complexes with mink ACE2, providing key insights
into how the Y453F mutation affects the binding of the S glycoprotein to
ACE28,9. However, the structure of Y453F trimeric S
glycoprotein in complex with mvACE2 has yet to be solved, and many
conformational stages remain to be elucidated. In this study, we
presented the cryo-EM structures of the trimeric S glycoprotein
containing the cluster 5 S1 mutations (Δ69-70, Y453F, D614G) complexed
with mvACE2 receptors in various conformational stages. Notably, our
study captured an intermediate step in which mvACE2 is bound to the RBD
of the S glycoprotein at a lower angle than previously
reported13,14,33, facilitating the further opening of
the RBD. Collectively, our results provide further structural insights
into the initial residue interactions between the S glycoprotein and
mvACE2, and the molecular mechanisms involved in the conformational
changes of the S glycoprotein upon binding to the host mvACE2 receptor.
2. Materials and Methods: