3.5. The conformational changes of mink S glycoprotein with
mvACE2 receptor
Herein, we performed a simultaneous structural comparison of
mvACE2-unbound and mvACE2-bound to spike at various RBD angles to
investigate the different conformational changes induced by binding.
Previous studies have reported that spike glycoprotein could only
accommodate ACE2 binding when its RBD is up at an angle of at least 50°
with respect to the horizontal plane of the S
glycoprotein14,33,39. However, this data shows that
about half of the mink S glycoprotein bound to one mvACE2 accommodated
the mvACE2 binding with the RBD at a lower angle than previously
reported13,14,33 (Fig. 2 and 6A).
Further structural comparison of our density map shows that the
mvACE2-free conformation had its RBD at 27.1° in relation to the
horizontal plane of mink S glycoprotein (Fig 2A). In comparison, one
mvACE2 and two mvACE2 bound spikes had their RBD rotate outward with an
up position at 62.8° and 69.2°, respectively (Fig 2C and 2D). The angle
at which mvACE2 is bound to the upRBD in our study is comparable to the
angles previously reported14,33,39 (Fig 6B and 6C).
The intermediate stage of the mvACE2-bound mink S glycoprotein with
downRBD captured was at an angle of 28.9°, similar to the angle of
mvACE2-free conformation and approximately 34°- 40° lower than when it
had up RBD (Fig. 6A). These findings challenge the previously reported
notion that ACE2 binding may only occur when the RBD is up at a high
angle as well as provide insight into the conformational changes that
are induced upon ACE2 binding.
4. Discussion:
Our in vitro binding studies demonstrate a weaker binding
affinity of the SD614G protein to the mvACE2 receptor
compared to the hACE2 receptor, and this suboptimal fitness in a new
host may be overcome by selecting virus with host-adaptive mutations.
Indeed, mutations were frequently observed in SARS-CoV-2 isolates
derived from infected mink26,41. We show that the
addition of the mink-associated S glycoprotein mutations Δ69-70 and
Y453F enhance binding to mvACE2 while having little impact on binding to
the hACE2 receptor, suggesting they are host-adaptive mutations to
American mink. The similarity in binding affinity between
SD614G+Y453F and SD614G+Δ69-70+Y453Fwith the mvACE2 receptor suggests that the Y453F change is the main
driver of the observed enhanced binding to mvACE2. Y453F has been shown
by others to enhance mustelid ACE2 usage8,9,42.
Residue 453 is located in the S glycoprotein RBD and interacts with
hACE2 receptor residue H34, which is a Y34 in mvACE2. To elucidate the
enhanced mvACE2 receptor binding mechanism, we solved the structure of
the American mink S glycoprotein trimer bound to the mvACE2 receptor.
As previously reported, the RBD was at a range of tilts and angles
regarding the horizontal plane of spike33. The general
consensus was that the RBD of the spike protein undergoes a
conformational shift in its RBD from an inactive ”down” state to an
active ”up” state at an angle of at least 50° to access the ACE2 of the
host cell14,33,39. Our study captured a novel
intermediate step in which the mvACE2 binds to mink S glycoprotein with
the RBD at a relatively lower angle than previously reported (Fig 4 and
6A). The simultaneous structural comparison suggests that the binding of
the mvACE2 receptor facilitates the further opening of the CTD1 of the S
glycoprotein. This would expose the fusion cleavage site of S2 in the
spike, triggering the release of the S1 subunit from the S glycoprotein
trimer43.
Overall, our structural analysis of the full trimeric mink S-mvACE2
complex is mainly in agreement with previously published
structures5-14 in its architecture. Additionally,
comparing the molecular interface between previously published mink S
RBD-mink ACE2 complex and our full trimeric mink S-mvACE2 complex when
RBD is up at >50° revealed comparable interacting
residues8,9 (Fig 6). This includes the enhanced
interaction between S F453 with mvACE2 Y34 via π-π stacking from the
Y453F substitution mutation in the mink S glycoprotein.
Further analysis of the mink S-mvACE2 complex revealed that the
interacting residues in the interface of the mink S and mvACE2 molecules
differ depending on the angle at which the RBD is bound to mvACE2 (Fig
5C and 6C). These results provide insights into the residues in the
spike trimer that are important for initiating the binding of the host
ACE2 receptor. For example, while we observed that S R403, K417, and
F486 may play a role in initiating the binding of the ACE2 receptor, we
did not note any interaction in these residues when the ACE2 is bound to
S at a higher angle. Similarly, we note that S Y505 plays a role in both
initiating and maintaining the binding of host ACE2 receptors. This is
congruent with previous reports that Y505 is a critical viral
determinant for specific recognition of ACE2 by SARS-CoV-2 RBD and why
many potent neutralizing antibodies interact with this specific
residue42,44,45. Overall, our sequence alignment
indicates that these interacting residues of the spike in downRBD
conformation are well-conserved across different variants except the
Y505H substitution in the Omicron BA.1 variant (Fig 3). It is reported
that while the Y505H mutation in Omicron BA.1 significantly reduced ACE2
binding, and other mutations in the RBD compensated for its decreased
binding affinity13,46.
The interactions between SARS-CoV-2 S glycoproteins and ACE2 receptors
are of prime interest due to the essential role it plays in species
specificity, transmission and pathogenesis. SARS-CoV-2 is now endemic in
humans and will give rise to periodic epidemics similar to that of
influenza A and B viruses and respiratory syncytial virus. Mutations
will continue to emerge in the S glycoprotein to escape host immunity
and/or optimize interactions with hACE2, and performing similar
structural studies on new variants is necessary for understanding the
disease and updating vaccines. Monitoring S glycoprotein variants for
expanded or altered species specificity will also help assess the risk
of zoonosis and reverse zoonosis.