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\begin{document}

\title{Structural basis of the American mink ACE2 binding by Y453F trimeric
spike glycoproteins of SARS-CoV-2}



\author[1]{Bo Liang}%
\author[1]{Hyunjun Ahn}%
\author[2]{Brenda M. Calderon}%
\author[2]{Xiaoyu Fan}%
\author[1]{Yunrong Gao}%
\author[1]{Natalie L. Horgan}%
\author[2]{Nannan Jiang}%
\author[1]{Dylan S. Blohm}%
\author[2]{Jaber Hossain}%
\author[1]{Nicole Wedad K. Rayyan}%
\author[2]{Sarah H. Osman}%
\author[2]{Xudong Lin}%
\author[2]{Michael Currier}%
\author[3]{John Steel}%
\author[2]{David E. Wentworth}%
\author[2]{Bin Zhou}%
\affil[1]{Emory University Department of Biochemistry}%
\affil[2]{Centers for Disease Control and Prevention}%
\affil[3]{National Center for Immunization and Respiratory Diseases}%


\vspace{-1em}



  
  \date{May 25, 2024}


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\selectlanguage{english}
\begin{abstract}
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the
host cell by binding to angiotensin-converting enzyme 2 (ACE2). While
evolutionarily conserved, ACE2 glycoproteins differ across various
species and differential interactions with Spike (S) glycoproteins of
SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to
SARS-CoV-2 outbreaks on multiple American mink ( \emph{Mustela vison})
farms during the pandemic and gave rise to mink-associated S
substitutions known for transmissibility between mink and zoonotic
transmission to humans. In this study, we used bio-layer interferometry
(BLI) to discern the differences in binding affinity between multiple
human and mink-derived S glycoproteins of SARS-CoV-2 and their
respective ACE2 glycoproteins. Further, we conducted a structural
analysis of a mink variant S glycoprotein and American mink ACE2
(mvACE2) using cryo-electron microscopy (cryo-EM), revealing four
distinct conformations. We discovered a novel intermediary conformation
where the mvACE2 glycoprotein is bound to the receptor-binding domain
(RBD) of the S glycoprotein in a ``down'' position, approximately 34\selectlanguage{ngerman}°
lower than previously reported ``up'' RBD. Finally, we compared residue
interactions in the S-ACE2 complex interface of S glycoprotein
conformations with varying RBD orientations. These findings provide
valuable insights into the molecular mechanisms of SARS-CoV-2 entry.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Structural basis of the American mink ACE2 binding by Y453F
trimeric spike glycoproteins of SARS-CoV-2}

Hyunjun Ahn\textsuperscript{1}\#, Brenda M.
Calderon\textsuperscript{2,3}\#, Xiaoyu Fan\textsuperscript{2,3}\#,
Yunrong Gao\textsuperscript{1}\#, Natalie L. Horgan\textsuperscript{1},
Nannan Jiang\textsuperscript{2,3}, Dylan S. Blohm\textsuperscript{1},
Jaber Hossain\textsuperscript{2,3}, Nicole Wedad K.
Rayyan\textsuperscript{1}, Sarah H. Osman\textsuperscript{2,4}, Xudong
Lin\textsuperscript{2,3}, Michael Currier\textsuperscript{2,3}, John
Steel\textsuperscript{3}, David E. Wentworth\textsuperscript{2,3}, Bin
Zhou\textsuperscript{2,3}*, Bo Liang\textsuperscript{1}*

\textsuperscript{1}Department of Biochemistry, Emory University School
of Medicine, Atlanta, GA, 30322 United States

\textsuperscript{2}COVID-19 Emergency Response, Centers for Disease
Control and Prevention, Atlanta, GA 30329

\textsuperscript{3}Influenza Division, National Center for Immunization
and Respiratory Diseases, Centers for Disease Control and Prevention,
Atlanta, GA 30329

\textsuperscript{4}Division of Laboratory Sciences, National Center for
Environmental Health, Centers for Disease Control and Prevention,
Chamblee, GA, 30341

\# These authors contributed equally to this study.

* Correspondence: bo.liang@emory.edu and bzhou@cdc.gov

\emph{Abstract} :

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the
host cell by binding to angiotensin-converting enzyme 2 (ACE2). While
evolutionarily conserved, ACE2 glycoproteins differ across various
species and differential interactions with Spike (S) glycoproteins of
SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to
SARS-CoV-2 outbreaks on multiple American mink (\emph{Mustela vison} )
farms during the pandemic and gave rise to mink-associated S
substitutions known for transmissibility between mink and zoonotic
transmission to humans. In this study, we used bio-layer interferometry
(BLI) to discern the differences in binding affinity between multiple
human and mink-derived S glycoproteins of SARS-CoV-2 and their
respective ACE2 glycoproteins. Further, we conducted a structural
analysis of a mink variant S glycoprotein and American mink ACE2
(mvACE2) using cryo-electron microscopy (cryo-EM), revealing four
distinct conformations. We discovered a novel intermediary conformation
where the mvACE2 glycoprotein is bound to the receptor-binding domain
(RBD) of the S glycoprotein in a ``down'' position, approximately 34\selectlanguage{ngerman}°
lower than previously reported ``up'' RBD. Finally, we compared residue
interactions in the S-ACE2 complex interface of S glycoprotein
conformations with varying RBD orientations. These findings provide
valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

\emph{}

\emph{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 2020\textsuperscript{1}. 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) glycoproteins\textsuperscript{2}. 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 therapeutics\textsuperscript{3,4}.

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 cells\textsuperscript{3,5}.
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 complex\textsuperscript{5-14}.

While \emph{Rhinolophus affinis}represent the natural reservoir of
precursors to SARS-CoV-2\textsuperscript{15}, multiple species, in
addition to humans, are susceptible to infection\textsuperscript{16-28}.
In April 2020, outbreaks of SARS-CoV-2 on American mink (\emph{Mustela
vison} ) farms were first reported in the Netherlands, with later
outbreaks observed in mink farms throughout Europe and North
America\textsuperscript{25,26,29,30}. The virus was transmitted to mink
by infected human workers and was capable of transmission among American
mink and back to humans\textsuperscript{31}. 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 (\selectlanguage{english}[?]69-70,
Y453F, D614G, I692V, M1229I) in the S
glycoprotein\textsuperscript{31,32}. 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
hACE2\textsuperscript{8,32}.

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
ACE2\textsuperscript{8,9}. 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 (\selectlanguage{greek}Δ\selectlanguage{english}69-70, 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
reported\textsuperscript{13,14,33}, 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.

\emph{2. Materials and Methods:}

\textbf{2.1.~Expression and purification of the S glycoprotein and ACE2}

SARS-CoV-2 S glycoprotein expression plasmids were constructed to encode
the ectodomain of S glycoprotein (residues 1--1208, with a mutated furin
cleavage site and two stabilizing proline substitutions K986P/V987P)
followed by a T4 foldon trimerization domain and a polyhistidine
purification tag, as described previously \textsuperscript{34}. For
functional assays, different constructs were designed to encode S with
mutations found in wild-type human viruses or viruses from American mink
and are referred to throughout the text as S\textsuperscript{D614G},
S\textsuperscript{D614G+Y453F}, and
S\selectlanguage{greek}\textsuperscript{D614G+Δ69-70+Y453F}\selectlanguage{english}. For structural analysis, a
SARS-CoV-2 S glycoprotein expression plasmid with four additional
stabilizing proline mutations (F817P, A892P, A899P,
A942P)\textsuperscript{35} was constructed and is referred to throughout
the text as mink S glycoprotein for simplicity.

ACE2 expression plasmids were also constructed as described previously
to encode the ectodomain of dimeric ACE2 followed by a human IgG1 Fc
purification tag, referred to throughout the text as hACE2-Fc and
mvACE2-Fc \textsuperscript{34}. Additionally, a monomeric mink ACE2
construct, where the IgG1 Fc purification tag was replaced with a
polyhistidine purification tag, was constructed for structure
determination and is referred to throughout the text as mvACE2. The
American mink ACE2 sequence was determined
previously\textsuperscript{36}.

Expi293F cells (Thermo Fisher, A14527) were cultured in Expi293
Expression Medium at 37\selectlanguage{ngerman}°C and 8\% CO\textsubscript{2} in the Orbi-Shaker
(Sigma, Z763438) at 130 rpm. The cells were transiently transfected at a
3.0 x 10\textsuperscript{6} cells/mL density using an Expifectamine 293
transfection kit (Thermofisher, A14524). For polyhistidine-tagged
proteins, the supernatant was harvested by centrifugation after 72 hours
and supplemented with 20 mM Imidazole, followed by affinity
chromatography with Ni\textsuperscript{2+}-NTA agarose beads (GoldBio,
H-350). The sample was washed three times with 5 column volumes (CV) of
wash buffer (30 mM Na\textsubscript{2}HPO\textsubscript{4} pH 7.4, 500
mM NaCl, 30 mM Imidazole) and then eluted three times with 5 CVs of
elution buffer (20 mM Na\textsubscript{2}HPO\textsubscript{4} pH 7.4,
500 mM NaCl, 500 mM Imidazole). For Fc-tagged proteins, the supernatant
was harvested by centrifugation after 72 hours, followed by affinity
chromatography using HiTrap protein A columns (Cytiva). The sample was
washed with 10 CVs of wash buffer (20 mM sodium phosphate, pH 7.4) and
eluted with 5 CVs of elution buffer (0.1 M citric acid, pH 3). Sample
collection tubes were pre-loaded with neutralization buffer (1M Tris
HCl, pH 9).

The eluted proteins were concentrated with 50-kDa or 100-kDa MWCO
centrifugal filters. Protein samples used for bio-layer interferometry
(BLI) assays were used after affinity chromatography. Protein samples
used for structure determination were further purified by size exclusion
chromatography using the Superose 6 increase 10/300 GL column (Cytiva)
in buffer (20 mM Tris pH 8.0, 200 mM NaCl). The peak fractions were
collected and verified through SDS-PAGE gel analysis. The samples were
concentrated and flash-frozen in liquid nitrogen and stored at -80°C.

\textbf{2.2. Bio-layer interferometry assay}

The binding affinity between the various trimeric S glycoproteins and
dimeric ACE2 receptors was evaluated using the Octet RED96 instrument at
30°C with a shaking speed of 1000 RPM (ForteBio), as described
previously\textsuperscript{37}. Anti-human IgG Fc biosensors (ForteBio)
were used. Following 10 minutes of pre-hydration of anti-human IgG Fc
biosensors and 1 minute of sensor check, 7.5 nM of hACE2-Fc or 15 nM of
mvACE2-Fc in 10X kinetic buffer (ForteBio) were loaded onto the surface
of anti-human IgG Fc biosensors for 5 minutes. After 1.5 minutes of
baseline equilibration, 5 minutes of association was conducted at 10 --
100 nM S glycoprotein, followed by 5 minutes of dissociation in the same
buffer used for baseline equilibration. For binding assays using
mvACE2-Fc, the association was conducted with 25 -- 200 nM S
glycoprotein. The data were corrected by subtracting the signal from the
reference sample, and a 1:1 binding model with the global fit was used
to determine equilibrium dissociation constants (Kd). Statistical
analysis was performed using GraphPad Prism 9 software. Two-way analysis
of variance (ANOVA) with Dunnett's multiple comparisons test was used to
compare the binding affinity (Kd) of S glycoprotein variants with
hACE2-Fc or mvACE2-Fc glycoproteins, and significance was defined as P
\textless{}0.05.

\textbf{2.3. Electron microscopy specimen preparation and data
acquisition}

To prepare cryo-EM samples, 3 \selectlanguage{greek}µ\selectlanguage{english}L of \textasciitilde{}1.5 mg/mL mink S
glycoprotein-mvACE2 complex (1:2.2 molar ratio S to mvACE2) were applied
to glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3). The
grids were blotted for 3 seconds at 100\% relative humidity and
flash-frozen in liquid ethane using a Vitrobot Mark IV system (Thermo
Fisher Scientific). The cryo grids were imaged using a Krios cryogenic
transmission electron microscope operated at 300 kV. The microscope was
equipped with a Gatan K3 camera with a slit width of 20 eV, and a total
of 11,392 micrographs were collected at 81,000X magnification. The image
was exposed for 4.0 seconds fractionated with 40 frames with an
accumulated dose of 49.98 e\textsuperscript{-}/\selectlanguage{ngerman}Å\textsuperscript{2}. The
pixel size was 1.11 Å with a defocus range of -0.75 to -1.75 \selectlanguage{greek}μ\selectlanguage{english}m.

\textbf{2.4. Cryo-EM data processing}

All data processing was conducted in CryoSPARC
v4.1.1\textsuperscript{38}. Patch motion correction and CTF estimation
were performed before manually curating exposures to exclude 1,746
micrographs with statistical outliers. 4,458,674 particles were
initially extracted via blob particle picking from the remaining 9,646
micrographs, followed by template particle picking. Extracted particles
underwent a series of 2D classifications and 3D classifications for
refinement. Local refinement was further performed with a mask covering
the American mink S glycoprotein RBD interacting with mvACE2. The full
workflow is summarized in Figure S1.

\textbf{2.5 Model building and visualization}

Initial model building of the different conformations of mink S
glycoprotein and the mvACE2 complex was performed in UCSF ChimeraX using
PDB 6ACJ and 7LWI for global mink S-mvACE2 complex and PDB 7F5R for mink
S glycoprotein RBD-mvACE2 complex after local refinement as a starting
model. Several rounds of refinement in Phenix and manual building in
Coot were performed until the final reliable models were obtained. Model
and map statistics are summarized in Supplementary Tables 1-2.

Figures were generated using UCSF ChimeraX and PyMol. UCSF ChimeraX was
used to measure the angles between the RBD and spike
protein\textsuperscript{33}. The ''Define'' function was used to
generate the horizontal plane of the American mink S glycoprotein and
long axis across the RBD. The ''angle'' function was used to calculate
the angles between the plane and the axis.

Cryo-EM reconstructions and atomic models generated in this study are
available at the Protein Data Base (PDB) under the following accession
codes: mink S glycoprotein alone (8T21), mink S glycoprotein bound to
one mvACE2 with downRBD (8T22) or upRBD (8TAZ), mink S glycoprotein
bound to two mvACE2 (8T20), and local refinement of the RBD-ACE2
interface of downRBD (8T25) and upRBD (8T23).

\emph{3. Results:}

\textbf{3.1. The binding of S glycoprotein homotrimers to hACE2-Fc and
mvACE2-Fc}

To identify potential differences in the binding affinity of S
glycoproteins to hACE2-Fc or mvACE2-Fc glycoproteins, we performed
bio-layer interferometry (BLI) assays. We determined the equilibrium
dissociation constants (Kd) for trimeric S stabilized by 2 proline
substitutions (K986P and V987P)\textsuperscript{35} and harboring either
the human (S\textsuperscript{D614G}) or American mink-associated
mutations (S\textsuperscript{D614G+Y453F} or
S\selectlanguage{greek}\textsuperscript{D614G+Δ69-70+Y453F}\selectlanguage{english}) from immobilized dimeric hACE2-Fc
or mvACE2-Fc receptors. The S\textsuperscript{D614G} was chosen as it
represents the predominant SARS-CoV-2 viruses circulating in humans when
the variants infecting American mink emerged. The trimeric S
glycoproteins S\textsuperscript{D614G}, S\textsuperscript{D614G+Y453F},
and S\selectlanguage{greek}\textsuperscript{D614G+Δ69-70+Y453F}\selectlanguage{english} all bound to dimeric hACE2-Fc
receptor with similar affinity, and no statistically significant
differences were observed (Fig. 1). However, the
S\textsuperscript{D614G} displayed a 5-fold reduction in affinity when
the hACE2-Fc was substituted with mvACE2-Fc in the assay. The affinity
of S for mvACE2-Fc was restored to S\textsuperscript{D614G}-hACE2-Fc
levels by the Y453F mutation in the spike, as both
S\textsuperscript{D614G+Y453F} and
S\selectlanguage{greek}\textsuperscript{D614G+Δ69-70+Y453F}\selectlanguage{english}showed a \textgreater{}4-fold
increase of affinity to mvACE2-Fc, compared to S\textsuperscript{D614G}
(p\textless{}0.0001). The \selectlanguage{greek}Δ\selectlanguage{english}69-70 deletion in NTD didn't further increase
the binding affinity to mvACE2-Fc beyond the effect of the Y453F RBD
mutation (Fig. 1).

\textbf{3.2. Cryo-EM structural determination}

To facilitate structure determination, we prepared a trimeric mink S
glycoprotein, harboring some of the mutations (\selectlanguage{greek}Δ\selectlanguage{english}69-70, Y453F, D614G)
found in mink cluster 5, with six proline substitutions that are
reported to have increased protein yields and stability in the prefusion
state\textsuperscript{35}. We mixed the mink S glycoprotein with
monomeric mvACE2 at a molar ratio of 1:2.2 and incubated it for 15
minutes before subjecting the complex to vitrification for EM analysis.

Cryo-EM analysis of the complex determined four distinctive species:
mink S glycoprotein alone (13.8\%), mink S glycoprotein bound to one
mvACE2 with downRBD (16.0\%) or upRBD (14.3\%), and mink S glycoprotein
bound to two mvACE2 in the upRBD position (55.8\%) (Fig. 2). After 3D
classification and refinement, apo-mink S glycoprotein was resolved at
3.60 \selectlanguage{ngerman}Å, while three conformational states of the mink S
glycoprotein-mvACE2 complex were captured and determined at 3.83 Å, 3.75
Å, and 3.36 Å. The RBDs were flexible with respect to the horizontal
plane of the spike protein, as shown by the 2D and 3D classification
analysis, capturing four different RBD angles: 27.1°, 28.9°, 62.8°, and
69.2° (Fig. S1F and 2).

Cryo-EM structural analysis of the mink S glycoprotein shows that its
overall organization of the trimer is similar to other variants,
including Alpha, Delta, Omicron, and previously published mink S
glycoprotein (Fig. 3). Our captured apo-mink S glycoprotein was
determined to have three receptor binding domains in the down position.
The RBDs of the mink S glycoprotein structures are less well-resolved,
most likely due to its flexibility in the region compared to the rest of
the protein chain. However, it is clear that the overall architecture of
the different S variants is conserved, which is consistent with the
conserved high affinity for hACE2 receptor.

\textbf{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
protein\textsuperscript{14,33,39}.

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 apart\textsuperscript{40} (Fig 4C). Structural analysis (Fig.
4C-F) revealed that the binding of the two molecules in the conformation
is mediated by \selectlanguage{greek}π-\selectlanguage{english}cation interaction between S R403 and mvACE2 Y34 and
between S Y505 and mvACE2 R393, \selectlanguage{greek}π-π \selectlanguage{english}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, \selectlanguage{greek}π-π \selectlanguage{english}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
reported\textsuperscript{8,9}and as will be demonstrated in the
following section.

\textbf{3.4 Structure of SARS-CoV-2 S glycoprotein trimer with upRBD
bound to mvACE2 receptor}

One of the three mvACE2-bound spike trimers captured had a strong
density for mvACE2 bound to two of the RBD at 69.2\selectlanguage{ngerman}°. After multiple
rounds of additional 2D and 3D classification analysis, we determined
the complex structure of mink S glycoprotein bound to two mvACE2 at 3.36
Å (Fig. 5A, S1B, and S1D). The local resolution of the protein interface
between the complex was low due to its flexibility in the region.
Nonetheless, the overall architecture of the mvACE2-mink S glycoprotein
complex was comparable to the previously published structure. To
visualize the key interaction at the protein interface between the
complex, we performed local refinement with the mask covering the
RBD-ACE2 interface, returning the improved resolution of the density map
at 3.82Å (Fig. 5B, S1G, and S1I). To analyze the binding interface
between the complex, we selected residues located less than 4.0 Å
apart\textsuperscript{40} (Fig 5C).

Overall, our structure largely agreed with the previous Y453F RBD-mink
ACE2 in interface residues\textsuperscript{8,9} (Fig. 5D-F). Notable
interactions include a salt bridge between mink S D405 with mvACE2 H354
and S E484 with mvACE2 H79; hydrogen bond between S T500 with mvACE2
Y41, S G502 with mvACE2 K353, and S Y505 with mvACE2 R393; \selectlanguage{greek}π-\selectlanguage{english}cation
interaction between S Y489 with mvACE2 K31; and \selectlanguage{greek}π-π \selectlanguage{english}stacking between S
F453 with mvACE2 Y34 and S Y505 with mvACE2 H354. F453 in S glycoprotein
is unique to the mink variant among other S variants (Fig. 3), and as
indicated in our structural and binding affinity studies, as well as
noted in previous studies\textsuperscript{8,9}, Y453F mink S
glycoprotein enhances the interaction with mvACE2 Y34.

\textbf{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\selectlanguage{ngerman}°
with respect to the horizontal plane of the S
glycoprotein\textsuperscript{14,33,39}. 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
reported\textsuperscript{13,14,33} (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 reported\textsuperscript{14,33,39} (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.

\emph{4. Discussion:}

Our \emph{in vitro} binding studies demonstrate a weaker binding
affinity of the S\textsuperscript{D614G} 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 mink\textsuperscript{26,41}. We show that the
addition of the mink-associated S glycoprotein mutations \selectlanguage{greek}Δ\selectlanguage{english}69-70 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
S\textsuperscript{D614G+Y453F} and
S\selectlanguage{greek}\textsuperscript{D614G+Δ69-70+Y453F}\selectlanguage{english}with 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 usage\textsuperscript{8,9,42}. 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 spike\textsuperscript{33}. 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\selectlanguage{ngerman}° to access the ACE2 of
the host cell\textsuperscript{14,33,39}. 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
trimer\textsuperscript{43}.

Overall, our structural analysis of the full trimeric mink S-mvACE2
complex is mainly in agreement with previously published
structures\textsuperscript{5-14} 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 \textgreater{}50° revealed comparable interacting
residues\textsuperscript{8,9} (Fig 6). This includes the enhanced
interaction between S F453 with mvACE2 Y34 via \selectlanguage{greek}π-π \selectlanguage{english}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
residue\textsuperscript{42,44,45}. 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 affinity\textsuperscript{13,46}.

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.

\textbf{ACKNOWLEDGMENTS}

We thank the assistance and services provided at the Robert P. Apkarian
Integrated Electron Microscopy Core (IEMC) at Emory University. This
research was partly supported by the National Cancer Institute's
National Cryo-EM Facility at the Frederick National Laboratory for
Cancer Research under contract 75N91019D00024. This project was
supported in part by an appointment to the Research Participation
Program at the Centers for Disease Control and Prevention, administered
by the Oak Ridge Institute for Science and Education through an
interagency agreement between the U.S. Department of Energy and CDC. The
findings and conclusions in this report are those of the authors and do
not necessarily represent the official position of the U.S. Centers for
Disease Control and Prevention or the Agency for Toxic Substances and
Disease Registry.

\textbf{CONFLICTS OF INTEREST}

The authors declare that there are no conflicts of interest.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1. Binding of S glycoprotein trimers to hACE2-Fc and
mvACE2-Fc receptors.} (A) Representative binding profiles of S
glycoprotein trimers to the hACE2-Fc receptor. The hACE2-Fc receptor was
immobilized to biosensors, and binding to S glycoprotein trimers
(S\textsuperscript{D614G}, S\textsuperscript{D614G+Y453F}, and
S\selectlanguage{greek}\textsuperscript{D614G+ Δ69-70+Y453F}\selectlanguage{english}) was measured by bio-layer
interferometry\textbf{.} (B) Representative binding profiles of S
glycoprotein trimers to dimeric mvACE2-Fc receptors. The mvACE2-Fc
receptor was immobilized to biosensors and binding to S glycoprotein
trimers (S\textsuperscript{D614G}, S\textsuperscript{D614G+Y453F}, and
S\selectlanguage{greek}\textsuperscript{D614G+ Δ69-70+Y453F}\selectlanguage{english}) was measured by bio-layer
interferometry (C) Comparison of binding affinity (Kd) measured by
bio-layer interferometry of S glycoprotein trimers to hACE2-Fc or
mvACE2-Fc receptors.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2. Electron density map of the mink ACE2-free and mvACE2
bound SARS-CoV-2 mink S glycoprotein.} Diagram illustrating the density
map of ACE2-free mink S glycoprotein {[}PDB: 8T21{]}, one mvACE2-bound
mink S glycoprotein {[}PDB: 8T22 and 8TAZ{]}, and two mvACE2-bound mink
S glycoproteins {[}PDB: 8T20{]}. The relative percent sample of each
population is 13.8\% (A), 16.0\% (B), 14.3\% (C), and 55.8\% (D). The
angle between the long axes of RBD and the horizontal plane is also
shown below each species.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3} . \textbf{Comparison of different \emph{apo} spike
glycoprotein variants.} Structural comparison of the \emph{apo-} mink S
glycoprotein determined in this study {[}PDB: 8T21{]} (A) with
previously reported \emph{apo-} mink S glycoprotein {[}PDB ID: 7LWI{]}
(B), Alpha S {[}PDB:7R13{]} (C), Delta S {[}PDB: 7SBK{]} (D), and
Omicron S {[}PDB: 7T9J{]} (E). The sequence alignment of the S
glycoproteins RBD is shown below, and the conserved residues are
indicated in black. Notable mutations in the mink S glycoprotein include
substitution mutation Y453F.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4. Cryo-EM structure of mvACE2 bound downRBD in
SARS-CoV-2 mink S glycoprotein.} (A) Cryo-EM map of downRBD spike
glycoprotein-mvACE2 complex at 3.75 \selectlanguage{ngerman}Å resolution. (B) Cryo-EM map of the
Mink S glycoprotein RBD with mvACE2 after local refinement at 3.62 Å
{[}PDB: 8T25{]} (C) Contacting residues less than 4 Å are labeled, and
lines indicate the interactions. Hydrogen bonds, contact, pi-cation, and
pi-pi stacking interactions are indicated by green, cyan, magenta, red,
and blue, respectively. (D-F) Close-up view of the RBD-ACE2 interface,
with key interacting residues labeled.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5. Cryo-EM structure of two mvACE2 bound to upRBD in
SARS-CoV-2 mink S glycoprotein.} (A) Cryo-EM map of upRBD mink S
glycoprotein-mvACE2 complex at 3.36 \selectlanguage{ngerman}Å. (B) Cryo-EM map of the Mink S
glycoprotein RBD with mvACE2 after local refinement at 3.82 Å {[}PDB:
8T23{]}. (C) Contacting residues located less than 4 Å are labeled, and
lines indicate the interactions. Hydrogen bonds, ionic, contact,
pi-cation, and pi-pi stacking interactions are indicated by green, cyan,
magenta, red, and blue, respectively. (D-F) Comparison of the close-up
view of the RBD-ACE2 interface between the interface of the trimeric
spike cryo-EM structure (top panel) and of the Y453F RBD-ACE in the
x-ray crystal structure {[}PDB: 7F5R{]} (bottom panel). The key
interacting residues are labeled.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6. Superimposition of the surface density of SARS-CoV-2
mink S glycoprotein bound to mvACE2.} (A) Comparison of previously
reported spike chain of the trimer bound to mvACE2 complex {[}green; PDB
ID: 6ACJ{]} with one mvACE2 bound S glycoprotein with downRBD {[}PDB ID:
8T22{]}, (B) one mvACE2-bound S glycoprotein with upRBD {[}PDB ID:
8TAZ{]}, (C) two mvACE2-bound S glycoprotein with upRBD {[}PDB ID:
8T20{]}.

\selectlanguage{english}
\FloatBarrier
\end{document}

