Received: 7 May 2021

Revised: 21 September 2021

Accepted: 8 November 2021

DOI: 10.1002/jev2.12170

RESEARCH ARTICLE

Millisecond dynamic of SARS-CoV- spike and its interaction with
ACE receptor and small extracellular vesicles
Keesiang Lim

Goro NishideAkiko Kobayashi

Takeshi Yoshida,Masaharu Hazawa,Takahiro Watanabe-Nakayama

Rikinari Hanayama,Toshio Ando

Richard W. Wong,
1

WPI-Nano Life Science Institute, Kanazawa
University, Kanazawa, Ishikawa, Japan

2

Division of Nano Life Science in the Graduate
School of Frontier Science Initiative, WISE
Program for Nano-Precision Medicine, Scienceand Technology, Kanazawa University, Kanazawa,
Ishikawa, Japan

3

Cell-Bionomics Research Unit, Institute for
Frontier Science Initiative (INFINITI), Kanazawa
University, Kanazawa, Ishikawa, Japan

4
Department of Immunology, Kanazawa
University Graduate School of Medical Sciences,
Kanazawa, Ishikawa, Japan

Correspondence
Keesiang Lim and Richard W. Wong, WPI-Nano
Life Science Institute, Kanazawa University,
Kakuma-machi, Kanazawa, Ishikawa 920–1192,
Japan.
Email: limkeesiang@staff.kanazawa-u.ac.jp andrwong@staff.kanazawa-u.ac.jp

Abstract
SARS-CoV-2 spike protein (S) binds to human angiotensin-converting enzyme 2(hACE2), allowing virus to dock on cell membrane follow by viral entry. Here, we
use high-speed atomic force microscopy (HS-AFM) for real-time visualization of S,
and its interaction with hACE2 and small extracellular vesicles (sEVs). Results show
conformational heterogeneity of S, flexibility of S stalk and receptor-binding domain(RBD), and pH/temperature-induced conformational change of S. S in an S-ACE2
complex appears as an all-RBD up conformation. The complex acquires a distinct
topology upon acidification. S and S2 subunit demonstrate different membrane docking mechanisms on sEVs. S-hACE2 interaction facilitates S to dock on sEVs, implying the feasibility of ACE2-expressing sEVs for viral neutralization. In contrary, S2
subunit docks on lipid layer and enters sEV using its fusion peptide, mimicking theviral entry scenario. Altogether, our study provides a platform that is suitable for realtime visualization of various entry inhibitors, neutralizing antibodies, and sEV-based
decoy in blocking viral entry.
Teaser: Comprehensive observation of SARS-CoV-2 spike and its interaction withreceptor ACE2 and sEV-based decoy in real time using HS-AFM.
K E YWOR D S
EV, exosomes, High-speed AFM, SARS-CoV-2 spike, ACE2



INTRODUCTION

Coronavirus Disease 2019 (COVID-19), a pandemic caused by a newly emerged beta-coronavirus (β-CoV), SARS-CoV-2, hasbrought devastating socioeconomic impacts to many countries (Guan et al., 2020; Huang et al., 2020; Sajidah et al., 2021). SARSCoV-2 is an enveloped, positive sense, single-stranded RNA virus. Like other enveloped viruses, SARS-CoV-2 entry requires
membrane fusion, and this process is orchestrated by a group of heavily glycosylated homotrimeric class I fusion proteins, known
as spike (S) proteins (Cai et al., 2020). An S protein consists of a globular head S1 subunit and a stalk-like S2 subunit. The S1subunit mediates host recognition whereas the S2 subunit manages membrane fusion. The S1 subunit possess four domains:
N-terminal domain (NTD), receptor-binding domain (RBD), and two structurally conserved subdomains (CTD1 or SD1, andCTD2 or SD2) (Henderson et al., 2020). Heptad repeats (HR1 and HR2) and the highly hydrophobic fusion peptide, importantcomponents for membrane fusion, are located in the S2 subunit (Epand, 2003; Tang et al., 2020). The S proteins of both SARSCoV-1 and SARS-CoV-2 bind to the same receptor, angiotensin-converting enzyme 2 (ACE2) (Hoffmann, Kleine-Weber, &Pöhlmann, 2020; Hoffmann, Kleine-Weber, Schroeder, et al., 2020; Walls et al., 2020; Yan et al., 2020). The RBD of SARS-CoV-2

This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided
the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.
© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles
J Extracell Vesicles. 2021;10:e12170.
https://doi.org/10.1002/jev2.12170

wileyonlinelibrary.com/journal/jev2

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S protein has higher binding affinity to ACE2 compared with the RBD of SARS-CoV-1 S protein (Wang et al., 2020). SARSCoV-2 S protein has two cleavage sites, S1/S2 and S2’. The S1/S2 site is cleaved by a host proprotein convertase, furin, converting S
protein into a metastable state (Fuentes-Prior, 2020). Upon receptor binding, either host surface serine proteases (transmembrane
serine protease 2, TMPRSS2) or host endosomal cysteine protease cathepsin B and L (CatB/L) cleaves S protein at the S2’ site
to remove the S1 subunit (Hoffmann, Kleine-Weber, & Pöhlmann, 2020; Hoffmann, Kleine-Weber, Schroeder et al., 2020). S1
subunit dissociation exposes the S2 subunit and triggers its rearrangement to complete membrane fusion (Cai et al., 2020).
Understanding the protein conformation and dynamic behaviour of S protein is crucial to block SARS-CoV-2 entry and expedite synthetic vaccine and target drug development. Several groups have reported that the S protein conformation is heterogeneous, as the protein exists in two different forms depending on the position of the RBD: a receptor-inaccessible form (closed
conformation; all RBDs down) and a receptor-accessible form (open conformation; at least one RBD up) (Cai et al., 2020; Wallset al., 2020; Wrapp et al., 2020; Wrobel et al., 2020; Xiong et al., 2020). Transition from a closed conformation to an open conformation depends on the interdomain interaction in the S1 subunit, in which the interaction can be perturbed by external factors
such as furin cleavage (Wrobel et al., 2020), pH (Zhou et al., 2020), temperature (Rath & Kumar, 2020), and receptor (ACE2)
binding (Lu et al., 2020; Zhou et al., 2020). Dynamic interdomain interactions confer the conformational plasticity of S protein,
and it is essential for SARS-CoV-2 to evade host immunity while retaining S protein fusion ability (Rey & Lok, 2018). Besides the
RBD, the conformational dynamics of S stalk have also been resolved by cryo-electron microscopy (cryo-EM) imaging. Turoňová
et al. (2020) and Ke et al. (2020) revealed that S stalk can bend and rotate on the viral surface. This dynamic property improves
receptor accessibility and hence enhances S protein attachment on the host surface. To date, the conformational dynamics of
S protein have been mainly dissected by cryo-EM imaging. Although cryo-EM is a powerful tool for structural analysis, manycryo-EM images are rather static snapshots. Furthermore, SARS-CoV-2 viruses used for cryo-EM imaging have to be fixed with
4% paraformaldehyde (Ke et al., 2020; Turoňová et al., 2020) and the conformational dynamics of S protein may therefore not be
fully recapitulated. Single-molecule fluorescence (Förster) resonance energy transfer (smFRET) has a high temporal resolution
in capturing the conformational dynamics of S protein (Lu et al., 2020); however, this approach only detects fluorescent signals.
High-speed atomic force microscopy (HS-AFM) is feasible for real-time observation of biomolecules with high spatiotemporal resolution at nano-scale range. We have performed biomolecular imaging using HS-AFM for structural characterization,observation of conformational dynamics, and to reveal the dynamic interactions of biomolecules and organelles (Ando, 2019;
Mohamed et al., 2017; Nishide et al., 2021). Recently, we used HS-AFM to investigate the conformational dynamics of influenza
A fusion protein, the hemagglutinin (Lim, Mohamed et al., 2020; Lim, Kodera et al., 2020). In this study, we used HS-AFM
to observe the native conformation and conformational dynamics of SARS-CoV-2 S ectodomain in different physicochemical
conditions, ACE2 binding, and interaction with small extracellular vesicles (sEVs). In addition, we also studied the interaction
between S2 subunit and sEVs.



MATERIALS AND METHODS

.

Materials

Recombinant proteins including SARS-CoV-2 S protein (S1+S2 ectodomain; 40589-V08B1), SARS-CoV-2 spike S2 subunit
ectodomain (40590-V08B), and human ACE2 ectodomain (10108-H08B) were purchased from Sino Biological Inc (Beijing,
China). These recombinant proteins were reconstituted in Milli-Q water at 0.25 mg/ml and then further diluted with appropriate buffers for HS-AFM recording.

.

Cell line

PC-9 cell line was purchased from the Immuno-Biological Laboratories Co (Gunma, Japan). HEC50B and A549 cell lines werepurchased from the Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan). HEC50B was cultured in
Minimum Essential Medium (MEM; Nacalai Tesque, Kyoto, Japan) with 10% foetal bovine serum (FBS; Life Technologies, CA,
USA) and 1% Penicillin-Streptomycin (Nacalai Tesque, Kyoto, Japan). A549 and PC-9 were maintained in Roswell Park Memorial
Institute (RPMI) 1640 culture medium (Gibco, MA, USA) supplemented with 10% FBS and 1% Penicillin-Streptomycin. All celllines were cultured at 37◦ C in a humidified CO2 incubator.

.

Preparation, purification, and characterization of sEV

sEVs of HEC50B, PC-9, and A549 were obtained by using the protocol mentioned in our previous study (Lim, Mohamed et al.,2020; Lim, Kodera et al., 2020). Briefly, cells were cultured for 4 days, and then cell-conditioned medium was subjected toLIM et al.

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sequential centrifugation to remove cells, debris and large EVs. Supernatant was then filtered through a 0.22 μm Millex-GV
filter (Merck Millipore, MA, USA) to produce a final pre-cleared cell conditioned medium, designated as the 10K sup. After that,sEVs in the 10K sup were purified by using a MagCapture Exosome Isolation Kit PS (Wako, Osaka, Japan) according to the manufacturer’s manual. The purified sEVs were then transferred into a dialysis membrane (3500 MWCO) for overnight dialysis in PBS(-). Concentration of sEV was measured by nanoparticle tracking analysis (NTA) using the NanoSIGHT LM10 (Malvern Panalytical, Malvern, UK). For detecting exosome markers, first, HEC50B, PC-9, and A549 cells were lysed with RIPA buffer. Then,cell lysates were collected by centrifugation at 15,000 × g for 30 min. Protein concentration of all samples were measured using
BCA protein assay kit (Thermo Fisher Scientific, MA, USA). 400 ng of sEV and cell lysate were used for SDS-PAGE and western
blotting. We follow the Minimal Information for Studies of Extracellular Vesicle 2018 (MISEV2018) guidelines (Théry et al., 2018)and have submitted all relevant data of our experiments (EV-TRACK ID: EV210256) to the EV-TRACK knowledgebase (Van
Deun et al., 2017).

.

Furin cleavage of S protein

Furin cleavage was carried out by incubating furin (P8077, NEB, MA, USA) with recombinant S proteins in a reaction buffer at
25◦ C for 1 h. The reaction buffer was made of 50 mM HEPES-KOH (pH7.4), 150 mM NaCl, 5 mM MgCl2 , and 2 mM CaCl2 . A
negative control (without furin treatment) was prepared. Samples were added with 4× SDS sample buffer and heated at 95 ◦ C
for 10 min to stop furin activity, then proceeded to SDS-PAGE and Western blotting.

.

SDS-PAGE and Western blotting

Protein samples were separated in 5%–20% SDS polyacrylamide gels and then transferred onto PVDF membranes. Blocking
was performed using 5% skim milk in PBST, followed by overnight primary antibody incubation at 4◦ C. Primary antibodies
used in this study were SARS-CoV-2 spike antibody (40591-MM42; Sino Biological Inc, Beijing, China), antibodies for exosome markers including CD9 (312102; BioLegend, CA, USA), CD63 (353039, BioLegend), CD81 (349502, BioLegend), TSG101(GTX118736, GeneTex, CA, USA) and VPS37B (HPA038217; ATLAS ANTIBODIES, Stockholm, Sweden), antibody for cellularmarker GAPDH (M171-3; MBL, Nagoya, Japan). After primary antibody incubation, PVDF blots were incubated with secondary
horseradish-conjugated anti-mouse (Cell Signaling Technology, MA, USA) or anti-rabbit (406401, BioLegend) at room temperature for an hour. Finally, Western blot images were developed using horseradish peroxidase (Millipore, MA, USA), and captured
using an image analyser LAS 4000 (Fujifilm, Tokyo, Japan).

.

Preparation of D protein structure and simulated HS-AFM images

The PDB files of S protein, spike S2 subunit, ACE2, and S protein-ACE2 complex were obtained from the RCSB PDB website(https://www.rcsb.org). PyMOL (The PyMOL Molecular Graphics System, Version 2.1.0; Schrödinger Inc, NY, USA) was used tovisualize the 3D protein structures and to label important domains of these proteins. BioAFMviewer software (WPI Nano LSI)
(Amyot & Flechsig, 2020) was used to generate simulated 3D HS-AFM images of these proteins.

.

Calculation of protein net charge and generation of surface electrostatic map

Total net charges of S protein, spike S2 subunit, and ACE2 at extracellular pH (7.4) were calculated using Prot Pi website (https://www.protpi.ch/). PDB2PQR (Dolinsky et al., 2004) and APBS (Adaptive Poisson-Boltzmann Solver) (Baker et al., 2001) analysis
were performed in the APBS-PDB2PQR webserver (https://server.poissonboltzmann.org/). After that, 3D surface electrostatic
maps of these proteins were generated using the PyMOL software.

.

Per-residue prediction of intrinsic disorder tendency

Prediction of the intrinsic disorder tendency in S protein and ACE2 were conducted using seven predictors including members of
the PONDR (Predictor of Natural Disordered Regions) family such as PONDR VLXT (Romero et al., 2001), PONDR VL3 (Penget al., 2005), PONDR VSL2 (Peng et al., 2006), PONDR FIT (Xue et al., 2010), IUPred for predicting long (≥30 residues) (Erdős& Dosztányi, 2020) and short IDPRs (<30 residues) (Mészáros et al., 2018), and PrDos (Ishida & Kinoshita, 2007). Residues withpredicted disordered propensity scores exceeding the threshold value of 0.5 are considered as intrinsically disordered residues. In

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contrast, residues with score in between 0.2 and 0.5 are considered flexible. Complete predicted percentage of intrinsic disordered(PPID) and complete predicted percent of flexibility (PPF) of these proteins were computed from outputs of seven predictors. In
addition, mean PPID and mean PPF were also calculated by averaging the outputs. Intrinsically disordered residues of S protein
and ACE2 were labelled in their respective 3D structure using the PyMOL software.

.

Generation of sharp cantilever tip by using electron-beam deposition (EBD)

BL-AC10DS-A2 cantilever was purchased from Olympus (Tokyo, Japan), and used as a scanning probe to image recombinant
proteins and sEVs. The cantilever has a spring constant (k) of 0.1 N/m and a resonance frequency (f) of 0.6 MHz in water (1.5 MHz
in air). The dimension of the cantilever is 9 μm (length), 2 μm (width), and 0.13 μm (thickness). EBD generates a long, sharp, andsmall apical radius tip on a cantilever to enhance image resolution. First, a cantilever was cleaned by UV/O3 and then in piranhasolution, containing sulfuric acid and hydrogen peroxide. Next, EBD was performed on the cantilever at 30 kV accelerating
voltage and 2 min of irradiation using a field emission scanning electron microscope, ELS-7500 (Tokyo, Japan).

.

HS-AFM visualization

HS-AFM images were obtained with our laboratory-build HS-AFM microscope, as previously described (Lim, Mohamed et al.,2020; Lim, Kodera et al., 2020). A laser beam (λ = 670 nm) passed through a 20× objective lens (CFI S Plan Fluor ELWD, Nikon,
Tokyo, Japan) and focused on an EBD-processed cantilever tip. The dynamic cantilever deflection was detected by sensing theposition of the laser beam reflected by the cantilever with a position-sensing two-segmented photodiode. A small tip-sample
loading force, which is crucial to protect sample structure, was generated by tuning the free oscillation amplitude of the cantilever(A0 ) to 1.5–2.5 nm and the set point to 80%–90% of the free amplitude. A glass stage glued with a stack of muscovite mica layers(∼0.1 mm thickness) was mounted on the HS-AFM scanner. The muscovite mica layers served as a substrate for adsorbing target
proteins and sEVs.
To scan native conformation of S protein, spike S2 subunit, and ACE2, recombinant proteins diluted with a scanning buffer(50 mM Tris HCl, 150 mM NaCl, pH7.4) were loaded on a suitable mica substrate and then incubated for 10 min. After that,
proteins were washed twice with the scanning buffer and proceeded to HS-AFM scanning. Bare mica was used as a substrate toadsorb S protein and spike S2 subunit whereas nickel (Ni2+ )-coated mica was used to adsorb ACE2. To prepare nickel (Ni2+ )coated mica, 3 μl of 0.5 M nickel chloride was added on bare mica and incubated for 15 min, then washed twice with the scanningbuffer.
To observe conformational dynamic of S protein at different pH, neutral scanning buffer (50 mM sodium acetate, pH 7.4) was
replaced with acidic scanning buffer (50 mM sodium acetate, pH 5) during HS-AFM scanning. For neutralization, the acidic
scanning buffer was then replaced with the neutral scanning buffer during the scanning.
To visualize the topology of S protein after exposed to different temperatures, S protein was first either incubated at room
temperature, 37◦ C, 60◦ C, or 90◦ C for 15 min. After that, the S protein was loaded on bare mica for HS-AFM observation undera neutral scanning buffer (50 mM Tris HCl, 150 mM NaCl, pH7.4).
For real-time observation of S protein-ACE2 interaction, empty nickel (Ni2+ )-coated mica was scanned to obtain backgroundimage. After that, ACE2 was added into chamber containing scanning buffer (50 mM sodium acetate pH7.4). Once the nickel(Ni2+ )-coated mica had been filled with ACE2, ACE2-containing scanning buffer was replaced with a plain scanning buffer
to remove excess ACE2. Lastly, S protein was added into chamber during HS-AFM scanning to observe the S protein-ACE2
interaction in a real-time fashion. To observe the conformational dynamic of S protein-ACE2 complex at low pH, the neutral
scanning buffer was replaced with an acidic scanning buffer (50 mM sodium acetate pH5). In the premix approach, S protein
and ACE2 were mixed and incubated in a scanning buffer (50 mM Tris HCl, 150 mM NaCl, pH7.4) for 30 min. After that, sample
were loaded on bare mica for HS-AFM scanning.
To visualize S protein- or S2 subunit-sEV interaction in a real-time manner, sEVs were first loaded on a poly-l-lysine (PLL)coated mica and incubated for 10 min. After that, sEVs were washed with a scanning buffer (50 mM Tris HCl, 150 mM NaCl,
pH7.4). Once an sEV had been found, either S protein or S2 subunit was added into chamber during HS-AFM scanning. For
premix experiment, S protein or S2 subunit and sEVs were incubated at room temperature for 3 h prior to HS-AFM imaging. A
negative control set (sEV only) was also prepared.
All HS-AFM scanning experiments were repeated at least three times to ensure the reproducibility of the experiments. Since thesame batches of recombinant proteins were used for HS-AFM scanning, repeated measurements were considered to be technicalreplicates.

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Force curve measurement

sEVs were incubated with S2 subunit at room temperature for 3 h before AFM imaging. Negative control (sEV only) was also prepared. Then, samples were immobilized on PLL-coated mica substrate, which was previously affixed on a 3-cm petri dish. Scanning buffer (50 mM Tris HCl, 150 mM NaCl, pH7.4) was used. A qp-BioAC CB3 cantilever (Nanosensors, Neuchatel, Switzerland)with a tip (tip radius < 10 nm, spring constant: 0.09 N/m, resonance frequency: 23–37 kHz in air) was equipped on a NanowizardII AFM scanner (JPK Instrument, Berlin, Germany) combined with an AxioObserver D1 inverted optical microscope (Carl Zeiss,
Germany) to image sEVs and to measure force–distance curve of sEV membrane. The AFM setting was identical for all force
mapping measurement. The imaging force was 0.1–1 nN, time per curve was 0.5 s, Z-length was 0.5 μm, and speed of curve acquisition was 1.0 μm/s. The optical lever sensitivity and the spring constant of the individual probes in liquid were obtained from theforce curve and the thermal noise measurements on the JPK SPM software. All images and force–distance curves (FDCs) wereanalysed using the JPK Data Processing software. Extend segment of each FDC was fitted to a Hertzian/Sneddon model modified
for a paraboloid AFM probe to obtain the Young’s modulus value. The Poisson ratio of sEVs were assumed to be constant, witha value of 0.5. Mean Young’s moduli between control group and S2 subunit-sEV group were compared using SPSS software.

.

Analysis of HS-AFM images

All HS-AFM images were processed by using the ImageJ software (https://imagej.nih.gov/ij/). The HS-AFM images were first
filtered by a fit polynomial filter (order for both x-and y-directions: 1) then by a low pass filter (Gaussian blur, Sigma radius: 2 nm)
to suppress noise and enhance image features. For some HS-AFM images, a Fast Fourier Transform (FFT) bandpass filter wasused to perform pixel filtration to remove both large and small structures in order to increase image details. A 3D surface plot
plugin in the ImageJ was used to generate 3D images of the recorded HS-AFM images. Besides image processing, parameterssuch as cross-sectional height, area, and circularity were also measured by using the same software. A circularity value of 1.0
represents a perfect circle. Values approaching zero indicate an increasingly elongated polygon. The circularity of S protein andsEV is given by 4πSL−2 , where L and S are the perimeter and the area surrounded by the object, respectively. The processed
images were converted to videos (AVI format). These videos were then arranged, edited, and compiled using Adobe Premiere
Pro CS6 software (Adobe).

.

Statistical analysis

Graphs for descriptive statistics were plotted using GraphPad Prism version 7 (GraphPad, CA, USA). Statistical analysis was
performed using SPSS version 22 (IBM Group, NY, USA). Comparative study of two-related samples was performed using the
Wilcoxon Signed-Rank test. Mann-Whitney U test and Kruskal-Wallis H test were conducted to compare independent samples.
The statistically significant level was set at p < 0.05 with a confidence interval of 95%. Sample size was determined based on ourearlier similar study (Lim, Mohamed et al., 2020; Lim, Kodera et al., 2020).



RESULTS

.

Native conformation of SARS-CoV- S protein as visualized by HS-AFM

Before HS-AFM imaging, we first obtained simulated HS-AFM images of S protein (Figure 1a), determined a suitable mica
substrate (Figure 1b and c), and predicted the potential intrinsically disordered region (IDR) in S protein (Figure 1d). To generate
simulated HS-AFM images of S protein in closed and open conformations, we downloaded PDB files of the cryo-EM structure of
S protein from the RCSB PDB data bank (PDB code, Closed: 6XR8 (Cai et al., 2020), Open 1-Up: 6VSB (Wrapp et al., 2020), Open
2-Up: 7A93 (Benton et al., 2020)). Simulation was performed using the BioAFMviewer software (Amyot & Flechsig, 2020). The
protein surface charge is a key parameter for S protein adsorption on a mica substrate. We calculated the net charge (Figure 1c)
and computed a surface electrostatic potential map (Figure 1b) (Baker et al., 2001; Dolinsky et al., 2004) for S protein, whichindicated that bare mica was feasible for S protein adsorption with sufficient mobility at pH 7.4 in the presence of salt (150 mM
NaCl). A single protein could comprise ordered and disordered domains. These structural properties could affect the protein
topology acquired by HS-AFM imaging. To determine the predisposition of IDR in S protein, we performed IDR prediction
using seven IDR predictors, including members of PONDR (PONDR VLXT, PONDR VL3, PONDR VSL2, PONDR FIT), the
IUPRED platform (IUPREDLong and IUPREDShort ), and PrDos. The output of the seven predictors together with the average ofthe outputs were illustrated in Figure 1d and Table S1-3. S was a highly ordered and flexible protein (mean PPID: 1.59%; mean PPF: of LIM et al.

F I G U R E  HS-AFM reveals three distinct conformations of SARS-CoV- S proteins. (a) Cryo-EM structures of S protein in close (PDB: 6XR8), 1-Up
(PDB: 6VSB), and 2-Up (PDB: 7A93) conformations as shown in their 3D structures. An explanation of the colours used is available in Figure S1A. These 3D
structures were used to simulate HS-AFM images (Sim HS-AFM). (b) PDB2PQR and APBS analysis of S protein were performed to generate surface
electrostatic maps at different salt concentrations and pH. (c) Titration curve computed from the Prot pi website using S protein amino acid sequence. The
isoelectric point and net charge at pH 7.4 are shown. (d) Prediction of intrinsic disorder region (IDR) predisposition in S protein. The predicted IDR regions
are labelled in red and yellow as shown in a 3D structure of S protein. (e) HS-AFM images showing a group or a single S protein at extracellular pH (scale bar,group, i: 25 nm, ii: 45 nm, iii: 60 nm; single, i: 12 nm, ii: 18 nm, iii: 20 nm). (f) 3D-surface plot of the S protein in three distinct conformations (* indicates RBD).
(g) Height histogram of the S protein

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31.89%). The S1 subunit had a higher mean PPID than S2 subunit (mean PPIDS1 vs. PPIDS2 : 2.53% vs. 0.39%) and S2 subunit wasmore flexible than S1 subunit (mean PPFS2 vs. PPFS1 : 41.41% vs. 23.85%). Two predicted IDRs were highlighted in red and yellow
in the 3D structure of S protein (Figure 1d). The first IDR (yellow) was located at the N-terminal domain (NTD; residues 16–24;
mean disorder score ± SD: 0.566 ± 0.038). The second IDR (red) was located at the furin cleavage site (residues 679–688; meandisorder score ± SD: 0.526 ± 0.016). Previous studies had also reported that a furin cleavage site exists as a disordered flexible
loop (Wrapp et al., 2020), and this structure favours furin binding. Direct visualization of the S protein in a physiological buffer
revealed a globular head and slender stalk topology (Figure 1e and f, Figure S1, B and C, and Movies S1 and S2), comparable
to the simulated HS-AFM images (Figure 1a). We managed to record both closed and open conformations of S protein using
HS-AFM (Figure 1e, Figure S1C, and Movie S2). Similarly, such conformational heterogeneity was also found in formalin-fixed
S protein (Figure S1D and Movie S3). 3D surface plots of both native and fixed S proteins demonstrate the hallmark crown-like
topology of S protein (Figure S1E). Western blotting results implied that S proteins were partially cleaved by innate furin, whichis expressed in insect cells, during recombinant protein synthesis (Figure S1F). Therefore, we managed to visualize S proteinsin an open conformation without pre-treating S protein with furin. Using HS-AFM, the conformation dynamics of S protein in
different conformations could be analysed at the single-molecule level. This idea is similar to single-cell analysis in which thesample pool is heterogeneous.

.

HS-AFM imaging reveals the dynamic behaviour of the stalk and RBD of S protein

Previous cryo-EM findings provided important clues about the dynamics of S stalk in tilting and rotating on the viral surface (Keet al., 2020; Turoňová et al., 2020; Yao et al., 2020), but the results only represented the dynamics of formalin-fixed S stalks. In
addition, the dynamics of S stalk have been depicted using molecular dynamic simulations (Ke et al., 2020; Turoňová et al., 2020).
Direct observation of S proteins on a SARS-CoV-2 virion using AFM is feasible (Kiss et al., 2021), yet the image resolution is
not ideal for analysis of the conformational dynamics of S stalk. HS-AFM provides a high spatiotemporal resolution in real-time
imaging that allows us to study the native conformational dynamics of S stalk without fixation. As predicted, the HS-AFM movie
demonstrated the highly dynamic S stalk and the less dynamic S head (Movie S2). The topology of the RBD loop resembled ashort tentacle protruding out of the S1 subunit head. We found that the RBD was dynamic but to a lesser extent than the S stalk.
This finding is in accordance with our flexibility prediction (mean PPFRBD vs. mean PPFS2 : 22.22% vs 41.41%) (Table S2 and S3).
We calculated the tilted angle relative to the axis that is perpendicular to the horizontal axis (Figure 2a and Figure S2A). The
horizontal axis could be considered as the viral membrane surface. Therefore, dynamic movement of S stalk during HS-AFM
scanning mimicked the dynamic tilting of S protein on the viral surface. We designated a negative tilted angle as left tilt, and apositive tilted angle as right tilt. Our results demonstrate that S stalk tilting was bidirectional. The tilted angle was able to reachclose to 90◦ , indicating that S protein could be tilted to touch the viral surface (Figure 2a). Besides tilting, we found that the S
stalk domain was able to extend or retract during HS-AFM scanning (Figure 2a and Figure S2A). This dynamic behaviour could
imply that S protein was able to move upward or downward on the viral surface. We further investigated the conformational
dynamics of the RBD loop (Figure 2b, and Figure S2, B and C). Similar to S stalk, the RBD loop tilted and extended or retracted
during HS-AFM scanning (Figure 2b and Figure S2, B and C). Intriguingly, the RBD loop intermittently disappeared during
HS-AFM scanning, when its loop length became zero (Figure S2, B and C). This finding suggested that interdomain interactionsin the S1 subunit may play a role in spontaneous conformation masking to hide the RBD. The tilting orientation of an S proteinwith two RBDs facing upwards was predominantly unidirectional tilting, which may be due to the repulsive force exerted when
two RBDs approaching each other.

.

Effect of pH and temperature on S protein conformation

Next, we investigated the effects of two physicochemical factors (pH and temperature) on S protein conformation. Low pH is
not the primary triggering factor for S protein to undergo fusogenic transition. Nevertheless, it could promote the evasion of
SARS-CoV-2 from neutralizing antibodies via antibody shedding (Zhou et al., 2020). Using HS-AFM, we aimed to study the
conformational dynamics of S protein induced by acidification followed by neutralization. For S protein in a closed conformation, we observed dissociation of the S head after acidification (Figure 2c, Figure S3A, and Movie S4, A-C). Following S headdissociation, the remaining S stalk underwent elongation at low pH (Figure 2ci and Movie S4A). In Figure 2 cii (see also Movie
S4B), we only visualized dissociation of the S head but no elongation of S stalk because we performed neutralization shortly after
dissociation of the S head. HS-AFM images in Figure S3B (see also Movie S4D) were captured in the middle of the acidification
process, the early phase of acidification was observed in another scanning area, so we were only able to observe the elongation
process. We found that elongation of S stalk was a reversible process upon neutralization. Multiple S stalks changed their conformation from globular to slender rods at low pH, then reverted back to their original globular conformation after neutralization.
For S protein in open conformation, dissociation of the S head did not occur. Instead, our data demonstrated that the frequency

 of LIM et al.

F I G U R E  Biophysical and morphological properties of the S protein. (a, b) HS-AFM scanning revealed a flexible S stalk (a) and RBD (b). Real-time
tilted angle and length change are shown in bar graphs and line graphs respectively (length ratio: length t = n /length t = 0 ). (c) Dissociation of the S head (i andii) and elongation of the S stalk (i) upon acidification (scale bar, i: 30 nm, ii: 25 nm). Real-time ratio changes in height (h t = n /h t = 0 ), area (A t = n /A t = 0 ), andcircularity (C t = n / C t = 0 ) were calculated and are illustrated in line graphs (blue: height, green dotted: area, red: circularity; blue area: pH 7.4, red area: pH5).
(d) Low pH induced significant reduction in S protein height. Data are presented as the mean ratio ± SEM of height before and after acidification, analysed byWilcoxon Signed-Rank test (n: 21; p = 0.0057, **p < 0.01). (e) Topology of S protein after exposed to different temperatures (scale bar, group, RT: 60 nm, 37◦ C:
75 nm, 60◦ C: 60 nm; single: RT and 37◦ C: 15 nm, 60◦ C: 20 nm). (f) Real-time height changes (Δ h = h t = n ˗ h t = 0 ) in the S protein exposed to different
temperatures. (g) High temperatures significantly increased the height of the S protein. Both height and circularity values are expressed in ratios relative to thesamples treated at RT. Data are presented as the mean ratio ± SEM (n, RT: 94, 37◦ C: 80, 60◦ C: 104; ****p < 0.0001, ns: no statistically significant)of RBD appearance was gradually reduced after exposure to low pH conditions for a prolonged period of time (Figure S3C andMovie S5). Surprisingly, the RBD was always visible after neutralization. The real-time height and circularity profiles of S protein in both the closed and open conformations appeared to follow a similar trend, with both height and circularity decreasing
during acidification, which was then reversed upon neutralization. We further analysed the effects of acidification on a groupof S proteins and found that the height but not the circularity of S protein was significantly reduced (Figure 2d). These findings

LIM et al.

 of 

implied that decreasing height was a general effect, whereas the loss of circularity only occurs in S proteins with conformationalchanges. The decrease in height may be explained in two ways. First, as mentioned above, dissociation of the S1 subunit reduced
S protein height. Second, protonation of S protein rendered its surface charge more positive (Figure 1b), strengthening its adsorption onto a bare mica surface via electrostatic interactions. Consequently, the height of S protein becomes reduced. This further
explains why S protein regains its height upon neutralization because its surface charge becomes less positive, thereby weakening
its adsorption onto a bare mica surface.
To investigate the temperature effect on S protein conformation, we heated S proteins at 37◦ C (S37◦ C ), 60◦ C (S60◦ C ), and 90◦ C(S90◦ C ) for 15 min before HS-AFM scanning. S protein incubated at room temperature (SRT ) was used as a control. S proteins were
found to be denatured at 90◦ C and failed to adsorb onto the mica surface (Figure S4A), whereas both S37◦ C and S60◦ C proteinsabundantly attached onto the mica surface (Figure 2e, Figure S4A, and Movie S6, C-F). Surprisingly, we found that S37◦ C proteins
forming a thread-like pattern (Figure S4, A and B, and Movie S6D), which was not observed with the SRT (Figure 2e, Figure S4A,
and Movie S, 6A and B) and S60◦ C (Figure 2e, Figure S4A, and Movie S6, E and F) proteins. Open conformations were found
for the S37◦ C proteins (Figure S4C, 37◦ C1 and 37◦ C2 , and Movie S7, E and F). Two RBDs protruded out from the S37◦ C protein
during scanning (Figure S4C, 37◦ C2 and Movie S7F). Although S60◦ C protein retained its spike conformation, most of the proteinsappeared in a closed conformation (Figure 2e, Figure S4C, and Movie S7, G, H, and I). We plotted the real-time height change
profile (ΔH; ΔH = Ht = n ˗ Ht = 0 ) of the three groups of S proteins (SRT , S37◦ C , and S60◦ C ) and found that S protein could havechanged its elasticity as the temperature increased (Figure 2f and Figure S4D). In addition, a high temperature could promote S
protein expansion as the height of S60◦ C was significantly higher than that of SRT and S37◦ C (Figure 2g).

.Conformation dynamics of S protein in the S-ACE interaction observed by HS-AFM in
real-time
The S protein–ACE2 interaction primes S protein for cleavage by host proteases, releasing S1 subunit and exposing the buried
fusion peptide, followed by the rearrangement of S2 subunit to orchestrate membrane fusion. Simulated HS-AFM images of
the ACE2 ectodomain were computed based on its 3D-model, which was generated by homology modelling using a cryo-EM
structure template (PDB: 6M18 (Yan et al., 2020)). ACE2 consists of a globular N-terminal peptidase domain (PD) and a taillike structure C-terminal collectrin-like domain (CLD) (Figure S5A). The net charge of ACE2 at pH 7.4 was highly negative(Z = ˗24.13), and negative charges were mainly distributed at the PD (Figure S5, B and C). IDR prediction analysis showed that
ACE2 is a highly ordered and highly flexible protein (PPID: 2.4%; PPF: 42.6%) (Table S4). The IDR region of ACE2 is located at
the CLD (Figure S5D). For ACE2 adsorption, a Ni2+ -coated mica substrate was used. Specific interaction between polyhistidine
tag in C-terminus of ACE2 and Ni2+ enabled ACE2 adsorbed on Ni2+ -coated mica substrate in an orientation similar to ACE2
on cell membrane. Therefore, majority of ACE2 appeared in their top view (globular) in HS-AFM imaging (Figure 3c, Figure S5,
E and F, and Movie S8). A small portion of ACE2 laterally adsorbed on the nickel (Ni2+ )-coated mica substrate due to its highnegatively charges at neutral pH. In this orientation, both PD and CLD are clearly visible on HS-AFM images (Figure S5Fi andMovie S8D). To visualize the interaction between S proteins and ACE2, we first computed simulated HS-AFM images of single-,
double-, and triple-ACE2-bound S proteins (Figure 3a) according to their respective cryo-EM models (Benton et al., 2020) (PDB:
single: 7A94; double: 7A97; triple: 7A98). The S protein–ACE2 interaction takes place at the PD region (blue) of ACE2 (Figure 3c).
We deleted the S2 subunit in the triple-ACE2-bound S protein structure (PDB: 7A98) to simulate a triple-ACE2-bound S1 subunit
complex, considering that S2 subunits might dissociate after S protein bound to the ACE2-coated mica surface during HS-AFM
scanning (Figure 3b). Visualization of the S–ACE2 interaction was carried out via two approaches: real-time observation andpremix incubation followed by HS-AFM scanning. The results of real-time observation of the S–ACE2 interaction are illustrated
in Figure 3d–g (see also Movie S9). We first added ACE2 protein into the chamber during HS-AFM scanning. ACE2 proteinsapproached and adhered to the nickel (Ni2+ )-coated mica substrate surface over time (Figure 3di and Movie S9, ACE2 Filling).
After ACE2 proteins had covered the substrate, excessive ACE2 was removed. Finally, we added S proteins into the chamber andobserved the interaction in real-time for about 13 min (800 s). S proteins were able to bind to the ACE2-coated mica surface,
but we did not detect any noticeable change in S protein conformation throughout the scan (Figure 3dii and Movie S9, S protein
added). ACE2-bound S proteins appeared to be either in a 2-Up or a 3-Up open conformation (Figure S6A). After observation of
the S–ACE2 interaction at pH 7.4, we then performed acidification to observe the conformational dynamics of ACE2-bound S
proteins (Figure 3diii and Movie S9, Acidification). We further focused on two areas, designated as “a” and “b”, to closely observe
the conformation dynamics of S protein starting from the addition of S protein to acidification of S protein (Figure 3e and Movie
S10). A noticeable rapid drop in both the height and area of ACE2-bound S proteins occurred (Figure 3f and Movie S10). We
did not observe dissociation of the S head throughout the acidification event (Movie S10). At higher magnification, we observed
several objects with similar topologies resembling five small balls connected to a central ball (Figure 3, Div and g, and Movie S11).
A schematic diagram is illustrated in Figure S6B to depict low pH-induced conformation transition in S-ACE2 complex. Since
the endosomal entry route of SARS-CoV-2 is mainly mediated by cysteine proteases (cathepsin B/L) (Hoffmann, Kleine-Weber,& Pöhlmann, 2020; Hoffmann, Kleine-Weber, Schroeder et al., 2020), we postulated that this structure could be the intermediate of 

LIM et al.

F I G U R E  Conformational dynamics of the S protein when interacting with monomeric ACE. (a) Cryo-EM structures of single- (PDB: 7A94),
double- (PDB: 7A97), and triple-ACE2 (PDB: 7A98)-bound S proteins are illustrated as molecular surface diagrams (blue: S protein; yellow: monomeric
ACE2). These structures were used to simulate their respective HS-AFM images. (b) Cryo-EM structure of the triple-ACE2-bound S protein (PDB: 7A98) wasedited by removing the S2 subunit to simulate the HS-AFM image of the triple-ACE2-bound S1 subunit. (c) Cryo-EM structure of the monomeric ACE2
ectodomain (PDB: 6M18) is depicted in the molecular surface diagram. The blue region indicates the binding site of RBD of S protein. (d) Real-timeobservation of the S protein–ACE2 interaction. HS-AFM observations were started at low magnification with ACE2 filling on the nickel (Ni2+ )-coated mica.
Extra ACE2 was removed, followed by the addition of S protein. Real-time interactions between S protein and ACE2 at pH 7.4 were observed for 800 s,
followed by acidification for another 800 s. Then, multiple sites were screened at mid magnification for the S protein–ACE2 complex under acidic conditions(scale bar, i: 150 nm, ii and iii: 250 nm, and iv: 125 nm). (e) Two areas (a and b) cropped from the figures in d(ii) and d(iii) delineated the real-time
conformational dynamics of S protein during interaction with ACE2 from neutral to acidic conditions (scale bar, Area a: 30 nm; Area b: 60 nm). (f) Real-timeheight and area changes in the S protein in E were measured and are presented as ratios relative to their respective initial height (ht = 0 ) and area (At = 0 ). The
results showed rapid declination in height and area upon acidification (blue area: pH 7.4, red area: pH 5). (g) S protein–ACE2 conformation after acidification
for 800 s as shown by high magnification HS-AFM scanning (scale bar: 13 nm). (h) HS-AFM images of the S protein–ACE2 complex after incubation of S
protein and ACE2 for 30 min at room temperature (scale bar, i: 25 nm; ii: 38 nm; iii: 25 nm). (i) HS-AFM observation revealed the ACE2-bound S protein witha dynamic S stalk (scale bar: 30 nm). Tilted angles were measured and are plotted in a bar graphLIM et al.

 of conformation of a triple-ACE2-bound S protein after acidification, priming the protease cleavage to reveal S2 subunit to complete
membrane fusion. In the premix experiment, we only observed the topology of the S–ACE2 complex at pH 7.4. We found that
S proteins predominantly appeared in a 3-Up open conformation (Figure 3h and Movie S12). In real-time observations, ACE2bound S protein could appear in a 3-Up open conformation, yet the contact point was beneath the S protein, making it appear
like a 2-Up open conformation. The S stalk remained mobile after the S protein bound to ACE2 (Figure 3i and Movie S13). This
result may imply that the mobility of S protein on the SARS-CoV-2 surface enables the virus to readily interact with the adjacentACE2, maximizing its attachment on the target cell surface.

.

S subunit disrupted the lipid bilayer stability of the sEVPreviously, we successfully recorded the real-time interaction between HA of human influenza A virus and sEVs under low pH
conditions using HS-AFM (Lim, Mohamed et al., 2020; Lim, Kodera et al., 2020). Our results clearly showed the insertion of
HA into the sEV lipid layer after fusogenic transition. Using a similar approach, we observed the interaction between the solubleS2 subunit and sEVs because the S2 subunit possesses the lipophilic fusion peptide. To observe the native conformation of thesoluble S2 subunit, we first prepared simulated HS-AFM images of the S2 subunit (Figure S7A) using two cryo-EM structures(PDB: 6ZWV for S2 homology modeling (Ke et al., 2020) and 6XRA (Cai et al., 2020)) and determined its net charge and surface
electrostatic potential at pH7.4 (Figure S7, B and C). Then, we scanned the S2 subunit on bare mica in a physiological scanningbuffer (50 mM Tris HCl, 150 mM NaCl, pH7.4). The S2 conformation was heterogeneous, either presenting as a slender rod ora bulky rod (Figure S7, D–F, and Movie S14). Such heterogeneity could be due to rearrangement of the S2 subunit (from thebulky rod to the slender rod). The S2 subunit actively interacted with sEVs (Figure S8 and S9, and Movie S15), and we grouped
these interactions into three categories: less than 1 s, less than 10 s, and more than 10 s (Figure 4a). A stable S2 subunit–sEV
interaction could promote insertion of the S2 subunit into the sEV (Figure 4b and c, Figure S10A, and Movie S16). The height
and length of the S2 subunit abruptly dropped to zero upon insertion (Figure 4d). We hypothesized that the S2 subunit insertion
could disrupt the stability of the exosomal layer, as we observed in our previous study (Lim, Mohamed et al., 2020; Lim, Kodera
et al., 2020). When we continued HS-AFM scanning over a longer period after S2 insertion, a declining trend in the height ofsEVs was observed (Figure 4e and Figure S10B). We also observed shrinkage of the sEVs followed by deformation of the sEVs
after two consecutive S2 subunit insertions (Figure S10, A and B, and Movie S16B). We further investigated this phenomenon by
incubating the S2 subunit with sEVs at two time points: 20 min and 2 h. We found that the S2 subunit was bound to sEVs at 20
min, and the sEV topology appeared rugged (Figure 4f and Movie S17B). After 2 h, sEVs appeared shrunken and completely losttheir spherical fluid-filled topology (Figure 4f and Movie S17C). AFM is a useful tool to measure various mechanical propertiesof biological samples (Ikai et al., 2018), and we used it to determine the effect of S2 subunit-sEV interaction on sEV membrane
stiffness. Result showed that sEV membrane stiffness was significantly reduced after sEVs interacted with S2 subunit (Figure
S10C), indicating that S2 subunit disrupted sEV membrane stability.

.

S protein potentially interacts with ACE receptor to stably dock on an sEVSeveral studies have revealed that extracellular vesicles (EV) secreted by host cells, express viral receptors that can neutralize
viruses to prevent viral entry (Conzelmann et al., 2020; de Carvalho et al., 2014; F. Cocozza et al., 2020). For example, T cells
secreted EV containing the HIV virus receptor, CD4+ , to neutralize HIV virus (de Carvalho et al., 2014). A recent study reported
that ACE2-expressing human epithelial cells, Calu-3 (lung) and Caco-2 (intestine), secreted ACE2-expressing EVs (F. Cocozzaet al., 2020). The cell line we used for sEV production, PC-9, is an ACE2-expressing cell line (Puray-Chavez et al., 2021). We
found that several S proteins could either transiently or stably attach onto the PC-9-derived sEV surface (Figure 5a, Figure S11,
A-C, and Movie S19). The transient S protein–sEV interaction showed a reversible interaction pattern (detaching and reattaching
to the same sEV) during HS-AFM scanning (Figure 5b and Movie S18). Multiple S proteins were able to bind to the same sEV(Figure 5, b, d, and e, Movie S18). To further emphasize the role of ACE2 in S protein-sEV interaction, by referring to dataobtained from the Cancer Cell Line Encyclopedia (CCLE, c-Bioportal), we isolated sEVs of high ACE2-expressing HEC50B andnegligible ACE2-expressing A549 (Figure S12A). Results show that HEC50B sEVs bound with several S proteins, but A549 sEVs
did not (Figure S12, B and C). The S protein–sEV interaction did not impose any effects on the S protein conformation because
no sudden change in height was observed throughout the interaction (Figure 5c). Although a recent study reported that theS1 subunit permeated and destabilized a neutral phospholipid bilayer (Asandei et al., 2020), our results revealed no prominent
declination in sEV height after a prolonged exposure to S proteins, suggesting that the S protein–sEV interaction did not disrupt
sEV membrane integrity (Figure 5f, Figure S12, B and C).

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LIM et al.

F I G U R E  Real-time interaction between the SARS-CoV- Spike S subunit and sEVs. (a-e) A representative HS-AFM visualization of S2
subunit-sEV interaction (n: 9). (a) Multiple S2 subunits interacted with sEVs for different lengths of time. The durations of the interactions were recorded andcategorized into three groups. Data are presented in bar graphs. (b) HS-AFM images show a S2 subunit (indicated by a white arrow) in contact with a sEV andsubsequent insertion into the sEV (scale bar: 100 nm). Graphs beneath the HS-AFM images indicate the distance between the S2 subunit and the sEV during
the interaction (red arrow: sEV, blue arrow: S2 subunit). (c) A kymograph shows a S2 subunit stably bound onto a sEV surface and eventually inserting into thesEV lipid layer. (d) Real-time height (h) and length (l) changes in the S2 subunit throughout the interaction with sEVs. The changes 6 s before contact (Free)and 6 s before insertion (Inserted) are illustrated in different line graphs. (e) Longer term observations of the S2 subunit–sEV interaction revealed that
insertion of the S2 subunit disrupted the integrity of the exosomal layer, as indicated by a declining trend in sEV height. (f) Disruption of the exosomal shape in
S2 subunit–sEV premixed samples. The S2 subunit was found to be attached to the sEV surface after incubation for 20 min and the sEV lost its spherical shapeafter 2 h of incubation with S2 subunit. A control sEV maintained a perfect spherical shape (scale bar: 50 nm)

LIM et al.

 of F I G U R E  Real-time interaction between S protein and an sEV. A representative HS-AFM visualization of S protein-sEV interaction (n: 6). (a)Multiple S proteins interacted with a sEV for different lengths of time. The durations of the interactions were recorded and categorized into four groups. Data
are presented in bar graphs. (b) The interaction between multiple S proteins or a single S protein and an sEV, as captured by HS-AFM. Graphs beneath the
HS-AFM images indicate the distance between the S protein and the sEV (scale bar: 50 nm, blue and orange arrows: S protein, black arrow: sEV). (c) Real-timeheight changes for the two S proteins during their interaction with the sEV. Orange areas indicate the height of S proteins when they were free from the sEV. (d)Close observation of the S protein–sEV interaction illustrates that the S protein slid along the sEV lipid layer (scale bar: 15 nm). S proteins are labelled witharrows. (e) A kymograph shows two S proteins touching (arrows) the sEV surface to form stable interactions, with both S proteins subsequently leaving(asterisk) the sEV. (f) Real-time height change demonstrate that the sEV height did not reduce after a long period of interaction with S protein. This resultshows that the S protein–sEV interaction did not disrupt the integrity of the sEV layer

DISCUSSION

HS-AFM has been proven to be a feasible methodology to study the native conformation and conformational dynamics of viralfusion proteins, and their interactions with sEVs (Lim, Mohamed, et al., 2020; Lim, Kodera et al., 2020). In this study, the SARSCoV-2 S protein topology captured using HS-AFM was comparable to previously reported cryo-EM structures. Besides the of 

LIM et al.

head and stalk, we managed to observe the native closed and open conformations of S protein at the single-molecule level. The
advantage of HS-AFM is that its high spatiotemporal resolution enabled us to visualize the highly dynamic behaviour of the S
stalk and RBD loop. The data acquired reflected the real-time active tilting, extension, or retraction of the S stalk or RBD during
HS-AFM scanning. These observations would be challenging to achieve by cryo-EM. Bioinformatic tools for IDR prediction cancomplement HS-AFM observations. For example, the outputs indicate that S protein is highly ordered or that S2 subunit is more
flexible than S1 subunit, in line with our observations from HS-AFM imaging. The same advantage of HS-AFM also enabled us
to track a continuous change in S protein structure during acidification follow by neutralization.
Our results demonstrate a complexity in the conformational dynamics of S protein in response to pH changes. Although S1
dissociation is mainly mediated by host proteases, spontaneous S1 dissociation in S protein with a closed conformation after
acidification may be possible. By contrast, low pH-induced conformation masking in S protein with an open conformation
supports a finding reported by Zhou et al. (2020). Technical limitations impeded our observations regarding the conformational
dynamics of S protein in response to a gradual change in temperature. Nonetheless, our current findings provide important
insights into the effects of temperature on S protein conformation. For example, at 37◦ C, S protein could become adhesive andthis property, together with the flexible S stalk, would allow SARS-CoV-2 viruses to form stable viral aggregates and spread to
adjacent tissue, or form respiratory aerosols or droplets. A high temperature significantly dampened SARS-CoV-2 infectivity(Riddell et al., 2020); however, S protein retained its conformation after heating to 60◦ C and the virus appeared to possess highglobal thermal stability (Kiss et al., 2021). Our findings regarding the S protein–ACE2 interaction indicate that this interaction
does not induce significant conformational change in S protein, but it could increase the propensity of the 2 Up- and 3 Up- open
conformations. These findings are consistent with results reported by other groups using cryo-EM (Henderson et al., 2020; Zhou
et al., 2020) and smFRET (Lu et al., 2020), suggesting that binding of single monomeric ACE2 disturbs interdomain interactions,and subsequently promotes the remaining RBDs to shift up. Future work is required to study the ACE2-bound S intermediatethat occurred after acidification.
ACE2 orientation on mica surface has to be as close as its orientation on cell surface in order to visualize the real scenario of S protein-ACE2 interaction during infection. Previously, Yang and coworkers used a bifunctional crosslinker, Nhydroxysuccinimide (NHS) and dimethylaminopropyl carbodiimide (EDC), to adsorb Fc-tagged recombinant ACE2 protein
on a gold-coated surface to measure S protein-ACE2 binding force using AFM (Yang et al., 2020). The NHS-EDC approachis not suitable for HS-AFM imaging of single ACE2 topology because it promotes protein aggregation (Valueva et al., 2020).
Ni2+ -histidine interaction has been shown to be applicable for orientating protein position on Ni2+ -coated substrates (Sumino
et al., 2017; Wasserberg et al., 2017; Yamamoto et al., 2010). Therefore, we used Ni2+ -coated mica substrate for ACE2 adsorption because our recombinant ACE2 has a poly-histidine tag at its C-terminus. ACE2 mostly appeared in a globular topology(PD) even though some displayed both globular (PD) and tail-like (CLD) topologies when ACE2 at low concentration. Likewise,
Yamamoto et al. demonstrated that his-tagged p97, a hexameric ATPase, bound to Ni2+ -NTA coated substrate via Ni2+ -histidine
interaction at neutral condition. The isoelectric point of p97 is close to the isoelectric point of ACE2 (Mori-Konya et al., 2009),
and hence it is negatively charged at neutral condition. Collectively, these findings indicate that Ni2+ -histidine interaction could
supersede non-specific electrostatic attraction.
EVs secreted by ACE2-expressing cell lines contain ACE2 receptors (F. Cocozza et al., 2020), and these EVs could work as
nano-decoys to bind with SARS-CoV-2 to block viral entry. sEVs also can be used to suppress hyperinflammation and to promote lung recovery in COVID-19 patients (Inal, 2020). Recently, three clinical trials of mesenchymal stem cells (MSCs)-derivedsEV based therapy (NCT04602442, NCT04276987, and NCT04798716) for COVID-19 treatment are currently ongoing. Nanoimaging of S protein–EV interaction could reflect the dynamic of S protein during its interaction with ACE2 on cell surface.
According to the International Society for Extracellular Vesicle (ISEV), EV is defined as a non-replicating lipid bilayer particle
released by cell (Théry et al., 2018). We used Tim4 isolation technique to acquire phosphatidylserine (PS)-enriched EVs from
HEC50B, PC-9, and A549 cell lines (Nakai et al., 2016). With this technique, we managed to harvest sEVs with exosome markers
including CD9, CD63, CD81, TSG101, and VPS37B (Figure S13), abiding the MISEV2018 guideline. S1 subunit in S protein mainly
mediates ACE2 receptor interaction and it does not disrupt sEV membrane integrity. However, after dissociation, free S1 subunit
becomes positively charge, allowing it to adsorb and permeabilize lipid bilayer (Asandei et al., 2020). We also found that the
S–sEV interaction was reversible. Further studies are now required to address whether SARS-CoV-2 could temporarily bind to
ACE2-expressing EV to evade antibody or to achieve systemic dissemination.
Besides S protein–ACE2 interaction, sEV has been used to visualize the S2 subunit-lipid bilayer interaction using HS-AFM.
Insertion of the S2 subunit into sEVs at pH 7.4 illustrates the second entry mechanism of SARS-CoV-2, in which the S2 subunit
orchestrates the fusion between SARS-CoV-2 viral membrane and the host plasma membrane to accomplish viral entry (Hoffmann, Kleine-Weber, & Pöhlmann, 2020; Hoffmann, Kleine-Weber, Schroeder et al., 2020). According to Ke et al., post-fusion
S proteins (without S1 subunit) were expressed on a SARS-CoV-2 virion (Ke et al., 2020). Also, two serine proteases secreted byneutrophils, neutrophil elastase (NE) and protease 3 (PR3), were found able to cleave S protein at S1/S2 site (Mustafa et al., 2021).
Additional works are needed to determine whether these soluble proteases could prematurely remove S1 subunit independentof ACE2 receptor and TMPRSS2. S protein without S1 subunit could favour fusion peptide in S2 subunit to interact with lipid
bilayer, potentially promoting immune evasion and expediting viral entry.

LIM et al.

 of In near future, HS-AFM can be considered for investigating the inhibitory effects of various approaches that targeting the
SARS-CoV-2 entry, either blocking S protein-ACE2 interaction or blocking S2 subunit-mediated membrane fusion. These
approaches including synthetic anti-S neutralizing antibodies (Copin et al., 2021), small-molecule inhibitors (Bojadzic et al.,2021), anti-S nanoparticles (Rao et al., 2020), or fusion inhibitor (Xia et al., 2020). Furthermore, HS-AFM nano-imaging of
antigen–antibody complex could provide important insights about antibody-dependent enhancement effect (ADE), as reported
previously (Asarnow et al., 2021; Liu et al., 2021). Altogether, we report that HS-AFM is an ideal tool for nanoscopic imaging toenhance our knowledge in SARS-CoV-2 S protein behaviour, allowing scientists to design effective countermeasures (sEV-basednano-decoys and vaccines) to manage COVID-19.
ACKNOWLED GEMENT S
We thank all members of the Richard Wong laboratory, especially to Mr. Hanbo Wang for helpful discussion. We also thank Prof.
Noriyuki Kodera for providing insightful HS-AFM advices in this study. This project was funded by grants from the MEXT/JSPS
KAKENHI (19K23841 (to K.L.), 20K16262 (to K.L.), 21H05744 (to R.W.), 21K19043 (to R.W.)) from MEXT Japan, the TDRP
grant (to K.L.) from WPI NanoLSI, the JST CREST (No. JPMJCR18H4 to R.H), from the Kobayashi International ScholarshipFoundation (to R.W.), the Shimadzu Science Foundation (to R.W.), the Takeda Science Foundation (to R.W).
AU T H O R C O N T R I B U T I O N S
Keesiang Lim and Richard Wong designed the study and wrote the manuscript. Keesiang Lim and Goro Nishide performed theexperiments and prepared figures. Takeshi Yoshida and Rikinari Hanayama prepared the small extracellular vesicles (exosomes).
Takahiro Watanabe-Nakayama performed AFM force–distance curve measurement. Akiko Kobayashi, Masaharu Hazawa andToshio Ando analysed the data. All authors read and approved the final manuscript. Richard Wong supervised the whole study.
DATA A N D M AT E R I A L S AVA I L A B I L I T Y
Not available.
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How to cite this article: Lim, K., Nishide, G., Yoshida, T., Watanabe-Nakayama, T., Kobayashi, A., Hazawa, M.,
Hanayama, R., Ando, T., & Wong, R. W. (2021). Millisecond dynamic of SARS-CoV-2 spike and its interaction with ACE2
receptor and small extracellular vesicles. Journal of Extracellular Vesicles, , e12170. https://doi.org/10.1002/jev2.12170