3.3 Structure of SARS-CoV-2 S glycoprotein trimer with downRBD
bound to mvACE2
In our cryo-EM analysis, we found that 30.3% of the species were
composed of the S glycoprotein bound to a single mvACE2 receptor (Fig.
2B and 2C). 16% of the single mvACE2 bound species had strong density
for mvACE2 bound to an RBD at an angle of 28.9° (”downRBD”) (Fig. 2B),
while the other14.3% (Fig. 2C) had strong density at an angle of 62.8°
(”upRBD”). The mvACE2 bound to the downRBD-spike (Fig. 2B) represents a
novel intermediary conformation that has not been previously captured.
Previous studies have suggested that the spike protein can only
accommodate ACE2 binding when its RBD is up at an angle of at least 50°
with respect to the horizontal plane of the spike
protein14,33,39.
To further elucidate the mechanism of this conformation, we resolved the
complex structure of mvACE2 bound to downRBD-spike at 3.75 Å (Fig. 4A,
S1C, and S1E). The structure was further improved using local refinement
covering the mink S glycoprotein RBD-mvACE2 interface, returning a 3.62
Å resolution of the density map (Fig. 4B, S1H, and S1J). To analyze the
binding interface between the complex, we selected residues located less
than 4.0 Å apart40 (Fig 4C). Structural analysis (Fig.
4C-F) revealed that the binding of the two molecules in the conformation
is mediated by π-cation interaction between S R403 and mvACE2 Y34 and
between S Y505 and mvACE2 R393, π-π stacking between S F486 and mvACE2
H79 and between S Y505 and mvACE2 H354, and ionic interaction between S
K417 and mvACE2 E30. Multiple sequence alignments of different S
glycoprotein variants show that these participating residues are mostly
well conserved (Fig. 3). Interestingly, we found that S F453 does not
form hydrophobic, π-π stacking or any contact interaction with the
mvACE2 Y34 (Fig. 4C and 4F), contrary to when the mvACE2 is bound to
upRBD of the spike, as it was previously reported8,9and as will be demonstrated in the following section.