Interfacial Water in the SARS Spike Protein: Investigating the Interaction with Human ACE2 Receptor and In Vitro Uptake in A549 Cells.

The severity of global pandemic due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has engaged the researchers and clinicians to find the key features triggering the viral infection to lung cells. By utilizing such crucial information, researchers and scientists try to combat the spread of the virus. Here, in this work, we performed in silico analysis of the protein-protein interactions between the receptor-binding domain (RBD) of the viral spike protein and the human angiotensin-converting enzyme 2 (hACE2) receptor to highlight the key alteration that happened from SARS-CoV to SARS-CoV-2. We analyzed and compared the molecular differences between spike proteins of the two viruses using various computational approaches such as binding affinity calculations, computational alanine, and molecular dynamics simulations. The binding affinity calculations showed that SARS-CoV-2 binds a little more firmly to the hACE2 receptor than SARS-CoV. The major finding obtained from molecular dynamics simulations was that the RBD-ACE2 interface is populated with water molecules and interacts strongly with both RBD and ACE2 interfacial residues during the simulation periods. The water-mediated hydrogen bond by the bridge water molecules is crucial for stabilizing the RBD and ACE2 domains. Near-ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) confirmed the presence of vapor and molecular water phases in the protein-protein interfacial domain, further validating the computationally predicted interfacial water molecules. In addition, we examined the role of interfacial water molecules in virus uptake by lung cell A549 by binding and maintaining the RBD/hACE2 complex at varying temperatures using nanourchins coated with spike proteins as pseudoviruses and fluorescence-activated cell sorting (FACS) as a quantitative approach. The structural and dynamical features presented here may serve as a guide for developing new drug molecules, vaccines, or antibodies to combat the COVID-19 pandemic.

Introduction

The rapid spread and high mortality rate (3–5%)[1] of the coronavirus (severe acute respiratory syndrome coronavirus-2, SARS-CoV-2) pose a serious global health emergency, taking more than 5.5 million lives all over the world (accessed date: January 07, 2022).[2,3] The new SARS-CoV-2 is characterized as a member of the bat coronavirus genome and closely related to the severe acute respiratory syndrome coronavirus (SARS-CoV).[3] SARS-CoV is a similar kind of virus that has already created a pandemic in the year 2002. Nevertheless, the magnitude of a fatality caused by SARS-CoV-2 surpasses all of the previous SARS-like viruses in terms of cost to human life and encouraged the desperate search for robust therapeutics.[4,5] Several vaccines and drugs currently exist that target SARS-CoV-2 infection. However, because of the evolving nature of the virus, the therapeutic efficacy of vaccines varies, and preventive measures are not fully effective. Both coronaviruses, SARS-CoV and SARS-CoV-2, use the homotrimeric spike glycoprotein (comprising S1 and S2 subunits) or the spike protein to bind the cellular receptors and induce the dissociation of S1 and S2 subunits. This also triggers a cascade of events such as the transition of S2 from a metastable perfusion state to a more stable postfusion state, the fusion between a cell and viral membrane, etc.[6,7] S1 subunit contains the receptor-binding domain (RBD) that binds to the peptidase domain (PD) of human angiotensin-converting enzyme 2 (ACE2).[8] Recent in vitro studies also indicate that the RBD of the S1 subunit plays a key functional role in the binding of SARS-CoV by ACE2.[9] Because binding to the ACE2 receptor triggers viral entry into the lung and marks the beginning of a viral life cycle, it is essential to determine how the binding affinity of SARS-CoV-2 differs from that of SARS-CoV. Experimental results reveal that SARS-CoV-2 has a slightly better binding affinity toward hACE2 than SARS-CoV.[10] The high binding affinity suggests that this coronavirus is evolved toward being a better binder to the same human ACE2 receptor. Thus, a comparison of both RBDs of SARS-CoV and SARS-CoV-2 and their stability in a varying biological environment (e.g., temperature, humidity, etc.) are of utmost importance. It might give a clue why SARS-CoV-2 is more infectious than SARS-CoV.

From the in silico perspective, it is still not fully understood why SARS-CoV-2 is a better binder than SARS-CoV-2 and the results are inconclusive.[11] Hence, an atomistic-level comparison is needed between these two virus spike proteins’ interaction with RBD that could shed fresh light on the role of spikes in viral stability and uptake. A recent study found that the hydrogen-bonding network and the hydrophobic interactions are major dominating forces that are responsible for enhanced binding in SARS-CoV-2.[12] Most of the studies focus on virus epidemiology and associated factors in addition to potent therapeutic designs against COVID-19. Very little attention is paid to the atomistic-level description and dynamics of the interfacial domain of the spike protein with the hACE2 receptor. The dynamic interactions between the RBD and hACE2 may reveal some crucial points that are not accessible from only the crystal structures.

In this work, we focus our study on the interfacial region of RBDs and hACE2 and provide an atomistic picture of the interactions. As a first step, we assessed the atomistic-level description of RBDs of the spike protein with the hACE2 receptor from the crystal structures. We identified the key contact residues dominated by molecular water bridges that are responsible for the binding of the RBD and hACE2.[11] We also critically analyze the differences in the interfacial residues of SARS-CoV and SARS-CoV-2. Further, the binding affinity of these two domains of SARS-CoV and SARS-CoV-2 was assessed. Next, all-atom molecular dynamics (MD) simulations of RBDs of the spike protein complexed with the human ACE2 receptor of SARS-CoV and SARS-CoV-2 are performed to extract the dynamics of these complexes. The importance of contact residues is also highlighted, and a thorough comparison was made between SARS-CoV and SARS-CoV-2. Our simulation results reveal that the interfacial water molecules play an important role in the binding of RBDs and the ACE2 domain, particularly the bridge water molecules connecting the two domains of the spike protein and hACE2. We experimentally confirmed the molecular and vapor phase of water in only SARS-CoV-2 spikes using sensitive near-ambient pressure X-ray photoelectron spectroscopy (NAP-XPS).[13] Taking a step ahead, to verify the role of water in virus infectivity, we design a pseudovirus model using spike -bearing gold nanoparticles (nanourchins) coated with SARS-CoV and SARS-CoV-2 spike proteins. We incubated these pseudoviruses at different temperatures to investigate the role of interfacial water in spike stability, hence infectivity to lung cells expressing ACE2 receptors to simulate the varying biological environment. Against the established notion that SARS-CoV-2 is more infectious, the experimental results indicate that SARS-CoV spike-coated pseudoviruses are taken up more in lung cells compared to SARS-Cov-2. The results suggest the possibility of additional factors influencing SARS-CoV-2 infectivity other than the spike protein. It will require further investigations to correlate other factors influencing viral infectivity.

Materials and Methods

Computational Modeling

The crystal structures of SARS-CoV-2-RBD/ACE2 (PDB ID: 6VW1) and SARS-CoV-RBD/ACE (PDB ID: 2AJF) are taken from the protein database and subjected to molecular dynamics simulations. The two protein–protein complexes contain some notable structural elements for which special care was taken while preparing the system, like N-glycosylation of various asparagine residues, disulfide bridges between various pairs of cysteine, and the presence of zinc finger and other amino acids, which require proper protonation states. The systems are prepared using the CHARMM-GUI web server,[14] and parameter files and equilibration files are taken from the server. All of the simulations were performed using software GROMACS version 2019.4[15] by the CHARMM-36 force field.[16] Each system is solvated with TIP3P[17] water, and KCl salt was added to neutralize the systems to a salt concentration of 150 mM. Each system is solvated in a rectangular box of dimensions L = 138 Å, L = 138 Å, and L = 138 Å. The distance is appropriate to avoid all finite-size effects due to long-range electrostatic interactions among neighboring simulation boxes. Simulations for both complexes are carried out at 310 K and 1 atm pressure. To treat long-range electrostatic interactions, the particle mesh Ewald (PME)[18] method is used with a cutoff of 12 Å, and to constrain hydrogen bonds, the LINCS algorithm is applied.[19] Each system is minimized with 5000 steps of the steepest descent algorithm to eliminate the bad contacts in the system. After minimization equilibration, simulations are performed in the NVT ensemble at 310 K. After NVT simulations, NPT equilibration is performed for 5 ns at 1 atm pressure, and the temperature is maintained at 310 K. During the equilibration period, the position restrained is applied to the heavy atoms of the protein. A velocity rescale thermostat and a Parrinello–Rahman Barostat are applied to maintain the temperature and pressure, respectively.[20] For the protein–protein binding affinity calculations, FoldX software is used.[21] Different visualizing and trajectory analysis software packages like UCSF Chimera[22] and Visual Molecular Dynamics (VMD)[23] were used, and QZyme Workbench, an in-house enzyme engineering platform, was used for all calculations performed and is discussed in the Results and Discussion.[24]

Release of SARS-Cov-2 Virus Mimicking Speech Droplet Generation

The experiments were conducted in a custom-made isolation room with a 1 m × 1 m × 1 m (L × B × H) boxed chamber with a negative pressure of −5 Pa. Airflow was marked with intrapulmonary smoke. The jet nebulizer was driven by air at a constant flow rate of 5 L/min, with the mask reservoir filled with sterile simulated lung fluid (Gamble’s solution, catalog number 1700-0800, Pickering Laboratories, Berlin, German) mixed with coronalike gold urchin nanoparticles and attached to the nebulizer aerosols was an outlet with condensation particle counter (CPC) and scanning mobility particle sizer (SMPS) equipment for measuring particle concentration and size distribution.[25] The multibranched spike-bearing gold nanoparticles, the as-called nanourchins (or nanoflowers alternatively), make a good shape/size mimetic nanodecoy study model. Considering the size of the SARS-Cov-2 virus in the range of 50–100 nm, we chose gold nanourchins of 90 nm.[26] We confirmed the size distribution of gold nanourchins before and after SARS-Cov-2 spike protein coating; however, no difference in sizes was observed, as shown in Figure S1, with the size distribution curve calculated by the intensity percentage. The ζ potentials of gold nanourchins were measured to be −24.7 and −33.4 mV, respectively, before and after spike protein coating. The polydispersity index (PdI) values recorded were 0.17 and 0.21, respectively, showing good shape stability of the gold nanourchins. An exhaled aerosol cloud was revealed as a jet plume, and the particle count was captured by CpC. The maximum dispersion distance of release particles (aerosols and droplets) through the nebulizer side vent was ∼1 m diagonally to the outlet setup for the particle measurement to estimate the particle concentration in the cloud.

ACE2/TMPRSS-Expressing A549 Cells, Spike Proteins, Fluorescent Tagging, and Conjugation with Coronalike Gold Nanourchins

A549 cells expressing ACE2/TMPRSS were commercially procured and cultured as per the manufacturer’s instructions (Creative Biogene Inc.). SARS-CoV and SARS-CoV-2 lyophilized spike proteins were purchased from commercial vendors (Creative Biomart, NY). The spike proteins fluorescently tagged with the fluorescein isothiocyanate (FITC) conjugation kit (Abcam Lightning-Link Cat# ab102884, Germany) were used to covalently link the dye. The gold-maleimide conjugation kit from Abcam (Cat# ab269903, Abcam, Germany) was used to covalently bind the fluorescently tagged spike protein with the coronalike gold nanoparticles (nanourchins, Cat# 797707, Sigma-Aldrich). The principle behind the conjugation chemistry is an activation reaction that occurs when a solution of the gold-maleimide nanoparticles is added to a solution of the thiol-activated molecule. Based on spectrophotometric measurements, we found that the spike protein distribution on the surface of SARS-CoV and SARS-CoV-2 nanoparticles corresponds to 46 ng/mL for SARS-CoV and 50 ng/mL for SARS-Cov-2.[27] We next divided the spike protein-conjugated gold nanourchins into four fractions to incubate at temperatures of 5, 20, 37, and 60 °C, respectively, to expose the nanourchins to A549 cells expressing TMPRSS/ACE2 receptors for the nanodecoy experiment.

Inhalation Exposure to A549 Lung Cells and Control Experiments with Nebulized Goldlike Particles

Air–liquid interface (ALI) culture models hold greater potential for the assessment of inhalation toxicity. While ALI setups have been one part of the effort to overcome the bottleneck of in vitro infection biology, parallel efforts are being made to develop identification methods.[28,29] For air–liquid interface mimicking lung exposure, an experimental cloud chamber (VITROCELL, Germany) was used to expose the A549 to the atmosphere. ALI cloud chambers are polycarbonate boxes with dimensions of 20 cm × 20 cm × 30 cm that hold two transwell plates (1 cm from the walls) containing cells grown at the air–liquid interface. Using this device, a cloud- or mistlike dispersion of a particulate dispersion was presented to respiratory endo/epithelial cells at the air–liquid interface in a transwell, where the upper chamber without any media represents the respiratory system’s air interface mimicking the alveolar sac and the bottom region of the transwell represents blood vessels (the liquid interface). We used approximately 1.2 × 107 nanourchins/mL in the mist and applied them to the A549 cells seeded at a density of 0.1 × 106 cells/well in complete cell culture media (Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penn/strep and 10% fetal bovine serum (FBS)). The porous transwell bottom allows cell culture media to reach at A549 cell monolayer in the upper chamber of the transwell. During a typical ALI exposure to the A549 confluent layer in a Petri dish, we nebulized the modified coronalike viral particles in simulated lung fluid for 90 s; i.e., the nebulizer is switched off at 90 s. The Petri dish containing nanourchins coated with spike fluorescent proteins stayed there for 5 min until no change in −Δf was observed (95% of the final count).[30] After waiting for another 3 min, cells were removed and placed into a humidity- and temperature-controlled incubation chamber (Memmert GmbH, constant climate chamber HPP110 series, Germany). We also performed control experiments with a longer waiting time of 10–15 min, but no change in −Δf was observed. These are optimized aerosol toxicity evaluation conditions as proposed by the Organization for Economic Co-operation and Development (OECD) and are lately regarded as a valid exposure strategy for in vitro toxicology.[31]

We performed control experiments also with nebulized simulated lung fluid with or without mixing nanourchin-like particles on filter paper, which were subjected to acid hydrolysis and inductively coupled plasma mass spectrometry (ICP-MS). For the analysis of the particle sample, measurements were performed using a quadrupole ICP mass spectrometer (iCAP Q, Thermo Fisher Scientific GmbH, Dreieich, Germany) equipped with a PFA ST Nebulizer, a quartz cyclonic spray chamber, and a 2.5 mm quartz injector (all from Thermo Fisher Scientific). The gas flows for the cool gas (Ar) and the auxiliary gas (Ar) were set to 14 and 0.65 L/min, respectively. Dwell times were set to 0.1.[32]

NAP-XPS Measurements of Interfacial Water on Spike Protein

Laboratory NAP-XPS measurements were done with an EnviroESCA (SPECS GmbH, Berlin, Germany). The monochromatic Al Kα X-ray source (1486.71 eV, 43 W, 14.5 kV) was separated from the measurement chamber by a silicon nitride window, and the PHOIBOS 150 NAP hemispherical energy analyzer combined with a 1D DLD detector was under ultrahigh vacuum (<1 × 10–8 mbar) due to a three-stage differential pumping system between the analysis section and analyzer. The entrance aperture (nozzle) had a diameter of 300 μm, and the usual working distance was one to two times the nozzle diameter.

The following three protein samples were analyzed: SARS-CoV (S1), SARS-CoV-2 (S2), and BSA (control). The individual proteins were drop-casted (50 μL, 50 ng/mL) on silicon substrates, which were used during this NAP-XPS study.[13] Designations S1 and S2 refer here to the sample ID, not spike protein subunit S1 or S2. The samples were fixed on a standard sample holder with conductive double-sided adhesive carbon tape and were measured as received at a pressure of 5 mbar.

All survey spectra were acquired in fixed analyzer transmission (FAT) mode at a pass energy of 100 eV, a step size of 1.0 eV, and a dwell time of 0.1 s. High-resolution O 1s, N 1s, and C 1s core-level spectra were recorded in fixed analyzer transmission (FAT) mode at a pass energy of 30 eV, a step size of 0.2 eV, and a dwell time of 0.1 s. The electron emission angle was 0°, and the source-to-analyzer angle was 55°. The binding energy scale of the instrument was calibrated according to ISO 15472 [ISO 15472:2010, surface chemical analysis—X-ray photoelectron spectrometers—calibration of energy scales, 2010]. The residual energy shift of the binding energy scale after environmental charge compensation by the gas was corrected for all spectra with respect to the C 1s photoemission line of aliphatic carbon at 285 eV.[33] Curve fitting of core-level spectra was done with SpecsLab Prodigy using a Gaussian/Lorentzian product function peak shape model in combination with the Shirley background. Generally, the full width at half-maximum (FWHM) was set as a free parameter but constrained to be the same for all peaks within the same core-level spectrum. All of the spectra were fitted with a minimum set of peak components.

Fluorescent-Activated Cell Sorting (FACS) Analysis to Investigate the Correlation of the Interfacial Water Layer on Spike Protein–ACE2/TMPRSS Interaction and Pseudovirus Internalization

To probe the role of interfacial water in the hydration state of the corona, we conjugated the spike protein from SARS-CoV-2 on gold nanourchins as synthetic viral nanodecoy and compared the results with the SARS spike protein, which are known for lesser interfacial water as the control in our computer simulation studies in the previous section (schematics below). In brief, we took 0.25 mg/mL spike protein and conjugated the protein on the gold NPs using a Thermo Fisher kit. In subsequent experiments, four Petri dishes were coated with these modified particles and left at different temperatures: at 4 °C in a refrigerator, room temperature at 20 °C, in a cell culture incubator at 37 °C and another at 60 °C while maintaining the constant relative humidity (RH) at each temperature. After 24 h, samples were collected and exposed to ACE2/TMPRSS-expressing A549 cells that were seeded in the Petri dish. After 72 h incubation, we analyzed the infectivity of these spike-bearing particles using FACS and confirmed with ICP-MS. Forward scatter height (FSH) against the side scatter area (SSA) was used as gating criteria to exclude the doublets in analysis by FloJo software, versions V.10.4.2 (BD Biosciences).

Inductively Coupled Plasma Mass Spectrometry

We followed our previous protocols to determine the coronalike gold nanourchins internalized by A549 cells with ACE2 receptors on the surface.[34,35] A total of 50,000 cells were seeded in six-well inserts (cat. no. 353180, Corning B.V., Netherlands; 0.4 μm pore size, 1.12 cm2). After 48 h of growth, the culture medium was changed; cells were washed once with phosphate-buffered saline (PBS), trypsinized, and centrifuged to collect the cell pellets in fresh media containing all supplements. The cell suspension was gently drop-coated into different Petri dishes incubated previously at 5 °C, 20 °C, 37 °C, and 60 °C, and after 24 h exposure, cells were analyzed for intracellular particle uptake by A549 cells expressing ACE2/TMPRSS receptors by inductively coupled plasma mass spectrometry (ICP-MS). It was implemented by washing the cells two times with PBS (each time 0.5 mL). This wash solution, as well as the original cell culture medium, was collected and subsequently microwave-digested to convert the particles into their ionic form. Later, the gold content was analyzed by ICP-MS.

Statistical Analysis

Data are shown as mean ± standard deviation. For SARS and SRAS-CoV-2 exposure, experiments in multiple wells were repeated with five triplicates in three independent biological experiments (N = 3). For the statistical analysis, a Mann–Whitney U-test was performed using Origin 9.1 software. *P < 0.05 was considered as significant; **P < 0.01; ***P < 0.001 (protocol for ICP-MS analysis).[36]

Results and Discussion

Structure and Energetics Comparison between SARS-CoV and SARS-CoV-2

Earlier published results highlighted the differences between the RBDs of two spike proteins. The spike protein of SARS-CoV and SARS-CoV-2 share 75% sequence similarity, and their structural folds are almost identical, whereas the RBDs have a sequence similarity of 73.7%.[3]Figure A,B shows the superposition of SARS-CoV and SARS-CoV-2, and in the inset, we highlight the key residues that have changed from SARS-CoV to SARS-CoV-2. In the crystal structures of SARS-CoV and SARS-CoV-2, significant protein–protein contacts can be seen, and we identify some of the key residues that are important for stabilization of the spike protein and hACE2 receptor. Among the 21 ACE2 residues that are interacting with two RBDs, 17 are found to be common residues. Most of the interacting residues of the ACE2 receptor are located near the N-terminal helix. The above analysis manifests that the surface area of the interfacial domain of ACE2 and RBD is almost the same. It is interesting to note that the interface of the RBD is enriched with tyrosine and glycine residues for SARS-CoV-2 (4 tyrosine and 3 glycine out of 14 residues) as well as SARS-CoV (6 tyrosine and 2 glycine out of 16 residues).

Figure 1

Structural alignment, crystal structure, and binding energy of the RBD of the spike protein with human ACE2 receptor of SARS-CoV and SARS-CoV-2. (A) For superposition, PDB ID (2AJF) and PDB ID (6VW1) are used for SARS-CoV and SARS-CoV-2. The inset shows the residues that are mutated from 2002 to 2019 in licorice form. (B) Crystal structure of SARS-CoV-2 (PDB ID: 6VW1) shown in (A). The green shaded area represents the space or the interfacial region in between the ACE2 and the RBD of the spike protein. (C) Computational alanine scanning results for SARS-CoV and SARS-CoV-2. Alanine scanning is performed on the residues of the RBD that are coming in contact with the ACE2 receptor (cutoff value 4.0 Å).

To see the effect of interfacial residues on the overall binding affinity with hACE2, we performed computational alanine scanning on these interfacial residues to determine the extent of the impact on the binding affinity due to alanine substitution. The crystal structure of SARS-CoV-2 has 13 hydrogen bonds between the RBD and the ACE2 domain. However, SARS-CoV has a fewer number of hydrogen bonds between the RBD and the ACE2 domain for SARS-CoV. It has only five hydrogen bonds between the two domains. As there are fewer hydrogen bonds in SARS-CoV, we expect that the RBD/ACE2 complex of SARS-CoV-2 is more stable than that of SARS-CoV.

Further, we calculate the binding free energy of the spike/hACE2 complex using the PRODIGY web server[37] as well as FoldX software.[21] The binding free energy is calculated on the crystal structures of the dimer, and the values are −10.8 and −11.7 kcal/mol for SARS-CoV and SARS-CoV-2, respectively. However, the FoldX calculations show binding energies of −4.49 and −9.87 kcal/mol for SARS-CoV and SARS-CoV-2, respectively. The values obtained from PRODIGY and FoldX are not in the same range. The PRODIGY trend suggests that SARS-CoV-2 binds more effectively to hACE2, whereas FoldX suggests that SARS-CoV evolved as a better binder to the human receptor.

The interfacial residues of the RBD are critical for stabilizing the protein–protein complex; we, therefore, performed computational alanine. Scanning on the RBD contact residues were performed to highlight the importance of those residues. We mutate the interfacial residues to alanine and then calculate the binding free energy with the ACE2 domain. Alanine scanning is performed to identify the residues that make a significant contribution to the binding affinity with hACE2. The result reveals that (Figure C, lower panel) most of the residues lower the binding affinity when mutated with alanine except N501 where a slight increase in binding affinity is observed for SARS-CoV-2. A decrease in the binding affinity suggests that both SARS-CoV-2 and SARS-CoV have optimized their RBD to bind with the human ACE2 receptor. This result points to the fact that SARS-CoV-2 optimized its interfacial residues in such a manner that binding to hACE2 is more enhanced. It is to be noted that Gln474 and Gly485 are inserted near the interfacial domain of RBD of SARS-CoV-2. Although these two residues are not directly interacting with ACE2, they form intramolecular hydrogen bonds to give stability to the loop. An antiviral immune response to viruses is often affected by accessory proteins encoded by viruses, and amino acid residues are crucial for it.[38]

Role of Interfacial Water Molecules

It is well known that water-mediated interactions are one of the major driving forces of protein folding and protein–drug recognition processes.[39−41] We observed that there is almost a 4–5 Å gap in between the SPIK/hACE2 protein–protein complex. There is a possibility that this interfacial domain or the gap region is hydrated with water molecules, and these interfacial water molecules might play a significant role in stabilizing the spike protein and the hACE2 receptor.[42] Indeed, from our simulations, we observed that water molecules gradually populate the interfacial domain of the spike protein and the hACE2 receptor. During the 100 ns simulation period, we observed that the interfacial domain forms a rich hydrogen-bonding network and stabilizes the overall protein–protein complex. From our simulations, we analyze the water molecules that come within 3.5 Å near the spike protein and hACE2 domain. Figure A–C represents the snapshot of water molecules that are 3.5 Å away from hACE2 and the RBD. The snapshots are shown here only for SARS-CoV-2, although similar results are also obtained for SARS-CoV.

Figure 2

Snapshots of interfacial water molecules from molecular dynamics simulations. (A) Water molecules within 3.5 Å from the hACE2 domain. (B) Water molecules within 3.5 Å from the RBD of the spike protein. (C) Bridge water molecules that are found at 3.5 Å from both the RBD of the spike protein and the hACE2 receptor. The above results are shown for SARS-CoV-2. A similar result is obtained also for SARS-CoV. (D) Interfacial water molecules near hACE2 calculated from the 100 ns MD simulation trajectory files. (E) Probability of interfacial water molecules that are near the hACE2 surface. (F) Interfacial water molecules near the RBD of the spike protein calculated from the 100 ns MD simulation trajectory files. (G) Probability of interfacial water molecules that are near the RBD surface. (H) Bridge water molecules calculated from simulation trajectory files for SARS-CoV and SARS-CoV-2. (I) Representation of one of the bridge water molecules that is hydrogen-bonded with both RBD and the hACE2 domain. Probability of hydrogen bonds between the water molecules and the interfacial domain of hACE2 and the RBDs (J) for the hACE2 receptor and (K) for RBD of the spike protein. Total interaction energy (Coulombic + van der Waals) between the water molecules and (L) hACE2 and (M) RBD.

The fluctuation in the number of water molecules in the interfacial region of the ACE2 domain shows high water content for SARS-CoV than that for SARS-CoV-2 (Figure D). The average numbers of water molecules near the ACE2 domain are 214.2 ± 8.68 and 242.7 ± 10.04 for SARS-CoV-2 and SARS-CoV, respectively. Though the ACE2 interface is almost the same for SARS-CoV and SARS-CoV-2, there is a higher number of water molecules near the ACE2 interfaces. The probability plot (Figure E) shows a clear difference in the number of water molecules between SARS-CoV and SARS-CoV-2. A similar result can be observed for the RBD of the spike protein. A greater number of water molecules can be seen near the SARS-CoV spike domain than those near the SARS-CoV-2 spike domain (Figure F,G). The high occupancy near the interfacial domain of SARS-CoV can be attributed to the fluctuation of the RBD and the hACE2 domain and a larger gap between these two domains.

Finally, we calculated the bridge water molecules that are simultaneously hydrogen-bonded with both the ACE2 domain and RBD at the same time. We calculate the number of water molecules that are simultaneously forming hydrogen bonds with RBD and the ACE2 domain. In Figure H, we display the number of bridge water molecules during 100 ns molecular dynamics simulations, and in Figure I, we show how bridge water molecule forms hydrogen bonds with RBD (Gln493) and the ACE2 (Glu35) domain at the same time. From the simulation trajectories, several multiwater bridge water molecules can be observed in RBD and the hACE2 domain. There is a slightly greater number of bridge water molecules (∼2) for SARS-CoV-2 than that for SARS-CoV. We expect that these bridge water molecules play a significant role in stabilizing the spike/hACE2 domain. Here, we want to highlight one key point that in the crystal structure (SARS-CoV-2) Gln493 is engaged in a hydrogen bond with Glu35, whereas from MD simulations, we observe that water molecules entered the interfacial region and connected these two residues.

Energy Contribution from Interfacial Water Molecules

To have an idea about how the RBD and ACE2 domains interact with water molecules, we calculate the water-mediated hydrogen bonds with RBD and hACE2 domains. In the case of hACE2, the interfacial residues make almost the same number of hydrogen bonds with the water molecules for SARS-CoV and SARS-CoV-2. This is expected as the ACE2 receptor is the same in both the viruses and small changes can be attributed to the fluctuation of the hACE2 domain. In Figure J, the probability of the number of hydrogen bonds with hACE2 is shown. The average numbers of hydrogen bonds formed between the water molecules and the hACE2 domain are 113.9 ± 6.4 and 113.2 ± 6.9 for SARS-CoV and SARS-CoV-2, respectively. Significant changes can be seen near RBD between SARS-CoV and SARS-CoV-2 (Figure K). The average numbers of hydrogen bonds that are formed between the interfacial residues of RBD and water molecules are 115.02 ± 6.4 and 109.5 ± 6.22 for SARS-CoV and SARS-CoV-2, respectively. The fewer number of hydrogen bonds for SARS-CoV-2 is the manifestation of mutation that occurred near the binding domain that enhances the hydrophobic nature in the interfacial domain.

In addition, we calculated the interfacial electrostatic and van der Waals interactions between the RBD and the ACE2 domain with water molecules (Figure L,M). Both viruses exhibit almost the same interaction energy between the water molecules and the hACE2 domain. Therefore, the surface topology of hACE2 behaves similarly in MD simulations for both viruses. In the RBDs of SARS-CoV-2 and SARS-CoV, a difference can be seen. Compared to SARS-CoV-2, SARS-CoV makes more hydrogen bonds with water molecules when interacting with the RBD. The increased water interaction with SARS-CoV’s RBD reduces direct interaction with the hACE2 receptor.

Role of Interfacial Water in Droplet Morphology: A Near-Ambient Pressure X-ray Photoelectron Spectroscopy (NAP-XPS) Analysis

Environmental and surface-sensitive NAP-XPS was used to detect the interfacial water (molecules) in the interfacial domain of the spike protein and hACE2 in the protein samples to verify the existence of computationally predicted interfacial water in SARS-CoV and SARS-CoV-2 spike proteins. SARS-CoV and SARS-Cov-2 spike proteins were mixed in the simulated lung fluid, nebulized, and coated on cleaned silicon wafers for NAP-XPS analysis. (NAP)-XPS is able to detect the surface chemistry and composition of the three investigated protein samples: BSA (control), S1, and S2. The surface compositions differ for the three samples, and almost no nitrogen was detected on the surface coated with the SARS-CoV-2 protein (S2) (cf. Table ). Oxygen and carbon 1s core-level spectra show significant differences between the three protein samples (Figure , atomic XPS spectra of different proteins are shown in Figures S2–S8, Supporting information).

Table 1

Elemental Compositions in Atomic Percent of BSA (Control), SARS-CoV-1 (S1), and SARS-CoV-2 (S2) Proteins as Calculated from NAP-XPS Survey Spectra Taken at 5 mbar

sample	C 1s (atom %)	N 1s (atom %)	O 1s (atom %)
BSA	64.1	10.9	25.0
SARS-CoV-1 (S1)	55.6	8.0	36.1
SARS-CoV-2 (S2)	63.2	1.8	35.0

Figure 3

NAP-XPS O 1s core-level spectra of BSA (top), SARS-CoV S1 (middle), and SARS-CoV S2 (bottom) proteins taken at 5 mbar.

Spike proteins from SARS-CoV (S1) and SARS-CoV-2 (S2) show more carbohydrate-like C 1s spectra. Moreover, SARS-CoV-2 protein S2 seems to have a significantly higher level of C–O species, as reflected by the O 1s and C 1s peak components located at 532.5 eV (–C) and 286.3 eV (–O) with corresponding total peak areas of 74 and 42%, respectively (cf. Table ).

Table 2

Peak Component Positions (eV) and Area Percentages of BSA (Control), SARS-CoV S1, and SARS-CoV S2 Proteins Obtained from the Peak Fits of the Respective O 1s Core-Level Spectra Taken at 5 mbar

sample	O=C and/or O=P (1) eV (%)	O–C and/or O–P (2) eV (%)	H–O–H (3) eV (%)
BSA	531.6 (71)	532.6 (29)
SARS-CoV S1	531.4 (56)	532.7 (44)
SARS-CoV-2 S1	530.8 (8)	532.5 (74)	533.6 (18)

Furthermore, the SARS-CoV-2 spike protein (S2) exhibits an additional third O 1s peak component located at 533.6 eV originating from interfacial water molecules (Figure and Table ).[43] This water-related component is completely absent in the BSA (control) as well as the SARS-CoV-1 spike protein (S1) (Figure and Table ).

As seen in the NAP-XPS spectra of the SARS-CoV-2 protein sample (S2), the specific water molecules associated with the spike protein might contribute not only to the infectivity but also to the droplet aerodynamics of the virus encapsulated in droplets emanating from speaking an infected person. The exhaled respiratory or speech air cloud from the infected person, which is initially humid and warmer, undergoes turbulent ambient air mixing in the atmosphere. It poses a challenge to the fundamental understanding of the transport process of the aerosols and droplets since the composition of the air cloud changes rapidly as it leaves the infected individual. Particularly, the aerosols transmitting via ambient air containing the SARS-CoV-2 spike protein having this vapor-phase water might retain infectivity in the shrinking dry core. Therefore, this result adds to our understanding of the role of the vapor phase of spike water contribution to viral stability predictions in drastically changing the external environment with existing knowledge of pH variation, salt composition, and protein coating around airborne viral transmission. The vapor phase of water might enable the airborne virus to survive in extreme conditions and work as a “reversible switch” to attain rapid infectivity once it makes appreciable biological contact to enter a new host.

In context with time-dependent droplet sedimentation and size variation due to cooling evaporation in speech droplets, the dominant solute osmotic-pressure effect of water is recently shown in theoretical studies;[44] however, no experimental verification exists until now. The water-mediated evaporation phenomenon may incredibly increase the viral air load by changing the sedimentation behaviors of the speech droplet. At a given time in a mid-size room, a maskless infected person may produce 10k virions in air at a steady state air load while speaking, which makes it prone to inhale by the people present in that room at an average of 2.5 virion/min. In functional and seasonal variations of air as encountered in laboratory/hospital bindings in winter and airline indoors, low relative humidity (RH) may exacerbate the airborne atmospheric loaf of virions, where again protein-bound water may have a critical role in droplet stability of the virus.

Role of Interfacial Water in Infectivity of SARS-CoV-2-Like Nanodecoys: Fluorescence-Activated Cell Sorting-Based Analysis of Intracellular Uptake in A549 Expressing the ACE2/TMPRSS Receptor

Such molecularly confined water in the vapor phase often exhibits anomalous dynamics, which have not been investigated concerning reactivation of viruses on confined surfaces and studied in relation to their infection potential.[39]

The water pockets detected in spike proteins and computationally observed interfacial water layers in the ACE2-spike protein could enormously add to our understanding of designing therapeutics and anti-infective surfaces to reduce the infection. The hydrophobic confinement of protein-associated water has been intensively investigated; however, there is no experimental verification of such proteins confined in biological surfaces that are mostly hydrophilic (humid) at body temperature (37 °C). We developed protocols to treat proteins at different temperatures keeping the humidity constant at 65% to explore further.

We used flow cytometry for rapid, qualitative, and quantification detection of intracellular uptake pseudonanodecoys coated with spike proteins covalently tagged with Cy555 fluorescent probes. As seen in the schematic in Figure , we develop a methodology to probe the role of water pockets in SARS-CoV and SARS-CoV-2 spike proteins. The water pockets in coronavirus spikes could eventually help in interfacial interaction between the lung cell ACE2 receptor and spike protein. We designed custom-made chambers where relative humidity can be kept constant, varying the temperature sequentially. As shown in the schematic in Figure , the fluorescent spike proteins covalently conjugated to gold nanourchins mimicking corona spikes were spray-coated on the cell l culture dishes and kept in an incubation chamber for 24 h at temperatures of 5, 20, 37, and 60 °C while keeping 70% RH for all four different temperature regimens.

Figure 4

To probe the role of interfacial water in the hydration state of the corona, we conjugated spike protein from SARS-CoV-2 on gold nanourchins as synthetic viral nanodecoys and compared the results with the SARS spike protein as the control, which are known for lesser interfacial water (Figures and 3). In brief, we took 0.25 mg/mL spike protein using a Thermo Fisher kit and conjugated the protein on gold NPs. Subsequently, five different Petri dishes were coated with these modified particles and left at 4 °C refrigerator, room temperature@20 °C, in cell culture incubator@37 °C and at @60 °C. After 24 h, samples were collected and ACE2/TMPRSS-expressing A549 cells were seeded in Petri dishes. After 72 h of incubation, we analyzed the infectivity of these spike-bearing particles using FACS and ICP-MS.

With increasing temperature and constant humidity, the relative abundance of water at high temperatures will be limited, and in these circumstances, we expect that these computationally detected water pockets help the virus to retain additional molecular water in equilibrium with the vapor phase of water as we saw in the previous section of NAP-XPS analysis. This could help the virus spike on coronalike pseudovirus (nanourchins) to infect and internalize into A549 cells expressing the ACE2 receptor. The upper panels in Figure A–H show the scatter plot demonstrating refraction or reflection of light at the interface between laser and fluorescent probe uptake by intracellular structures on the y-axis and forward scatter area on the x-axis as a function of the cell size. The control experiment was performed as a routine culture without exposing any nanodecoys with A549 cells (expressing ACE2/TMPRS receptor), and the results are shown in Figure S9.

Figure 5

Receptor-mediated uptake analysis of nanourchins in TMPRSS/ACE2-expressing A549 cells using FACS. Scatter plots (upper panel) and histograms (lower panel) of a population of A549 cells internalizing SARS-CoV and SARS-CoV-2 spike proteins, respectively, incubated at (A, B) 5 °C, (C, D) 20 °C, (E, F) 37 °C, and (G, H) 60 °C.

In the histograms of lower panels in the same figure, the measured values are directly tabulated across a set of channels or bins. The figure caption therein represents different spike proteins incubated and taken up by A549 cells. An overlay of control cells (without stain) with two categories of spike protein-internalizing cells assisted us in precisely isolating the exact number of cells internalizing the coronalike nanoparticle as a function of temperature-induced water loss recovery due to the internal water pocket detected by computation simulations. ICP-MS analysis corroborated the FACS data for the uptake of gold nanourchins as pseudovirus decoys, as shown in Figure A. Unstained isotype control was overlaid on PE-Cy555-internalizing A549 cells to relate the percent cell count (Figure B) and mean cell count (Figure C), showing a shift in intensity compared to the control. The overlay of unstained cells as a negative population here onto stained cells easily allows differentiating to percent population with coronalike particle uptake. These results indicate that despite having a vapor phase of water in SARS-CoV-2, infectivity is significantly lower compared to the SARS-CoV spike protein (Student’s t-test, p = 0.00001 at 5 °C; p = 0.0002 for 20 and 37 °C; 0.03 for 60 °C) irrespective of humidity and temperature variations. Considering the current pandemic, it looks unconventional; however, the low infectivity of SARS-CoV-2 Spike compared to that of SARS-CoV Spike could be due to the vapor phase of water, which might help virus transmission (e.g., vaporize faster) keeping the spike residue intact with host RBD interaction, but it did not assist in interaction with lung cells with changing environmental conditions (e.g., temperature). Proteins with hydrophobic and vapor-phase water components are known to transform from liquid to gaseous phase rapidly while maintaining significant structural memory.[45] Further looking at the effect of reduction in infection potential as a function of temperature in SARS-CoV-2, we see a significant decrease (Student’s t-test, p value = 0.0001 comparing 20 and 37 °C vs 60 °C, as shown in Figure B,C) in virus entry into A549 cells of up to 85-fold. This strategy could be useful in designing the surfaces or physical treatments for viruses-infected surfaces to reduce their infectivity while contacting them.

Figure 6

Quantitative analysis for uptake of pseudonanodecoys. (A) Results from ICP-MS, (B) PE-Cy555 internalized percent cell count, and (C) mean cell count, showing a shift in intensity compared to the control.

Figure presents the single-parameter histogram to differentiate the population of A549 cells with fluorescent pseudovirus uptake compared with control samples. The figures show the histograms of A549 cells exposed to PE_Cy555 conjugated with Sars-CoV and Sars-CoV-2 proteins at different temperatures (see Figures and 7 legends and the single-cell counts from FACS data in Figure S9 for details). Figure (A–C) exhibits the experiments performed at 5 °C, (D–F) at 20°C, (G–I) at 37°C, and (J–L) at 60°C. The first histogram shows the control without the PE_Cy555 treatment (top row). In the second and third row pictures, the red histograms represent the control and the green histograms with orange and blue represent the PE_Cy555-treated cells in triplicate. The shift in the histogram from the control denotes the cells that have uptaken the PE_Cy555-conjugated SARS-CoV and Sars-CoV-2 proteins. Comparing SARS-CoV and SARS-CoV-2 in the histograms in Figure and the supporting table in Figure S10, there is a clear trend of higher uptake of nanodecoys with SARS-CoV coating than that with SARS-Cov-2 spike protein coating irrespective of temperature-mediated spike protein alternations. The spike–ACE2 binding energy calculated computationally with FOLDX is high for SARS-CoV compared to that for SARS-Cov-2, which could be a determining factor herein for high binding and uptake of pseudoviruses coated with spike proteins. These results add an important point that virus-contaminated substrates may be tuned via temperature- and humidity-controlled protocols to bring down the viral infectivity by neutralizing the virus spike-based host–pathogen interactions.

Figure 7

Relative mode (control, SARS-CoV, and SARS-Cov-2) presentation of overlay FACS data. The count value given here is the number of single cells from the control histogram (top row, three repeat specimens with three colors), SARS-CoV (middle row, pink denotes control and the other three colors denote repeat specimens in triplicate), SARS-CoV-2 (bottom row, pink denoted control and the other three colors denote repeat specimens in triplicate). (A–C) 5 °C, (D–F), 20 °C, (G–I), 37 °C, and (J–L) 60 °C.

Conclusions

In summary, we elucidate the similarities and differences between SARS-CoV and SARS-CoV-2 by molecular dynamics simulation approaches. From the structural point of view, the RBD of the spike protein shares a significant similarity in terms of the three-dimensional structure and the fold. However, significant differences are observed in how these two viruses bind to the hACE2 receptor. We found that the improvement in hydrophobic contact that leads to enhanced van der Waals interactions between the RBD and the hACE2 receptor affects the high binding affinity for SARS-CoV-2. The most important information we found from the study is the involvement of water molecules in the interfacial domain of the RBD and ACE2 receptors. We observed computationally and experimentally using environmental-sensitive NAP-XPS that the interfacial domain for both SARS-CoV and SARS-CoV-2 is spontaneously hydrated and a significant number of water molecules populate the gap between RBD and the ACE2 domain. It is found that bridge water molecules play a significant role in stabilizing the protein–protein binary complex.[46] This kind of water-mediated interaction for SARS-CoV and SARS-CoV-2 was not observed in the previous study. Our results here demonstrate the role of interfacial water in the infectivity of the spike-bearing corona and may be useful in developing effective antiviral surfaces and therapeutics to prevent future pandemics by mitigating virus transmission and infection. In comparison with SARS-CoV-2, the alanine mutation does not reduce the binding affinity for all residues of SARS-CoV, which could explain the higher infectivity of these in vitro models we developed here. The present study unravels the many structural and dynamical features of this protein–protein dimer complex that help us to understand how this virus interacts with the human cell.