Solene Lecot

Yann Chevolot

Magali Phaner-Goutorbe

Christelle Yeromonahos
email: christelle.yeromonahos@ec-lyon.fr

Solène Lecot

Yann Chevolot §$

The Curious Binding Energy Increase between the Receptor-Binding Domain of the SARS-CoV-2 Spike Protein and the Angiotensin-Converting Enzyme 2 Adsorbed on a Silane Monolayer, from Molecular Dynamics Simulations

published or not. The documents may come

Introduction

Antigenic tests are widely used as diagnosis tools in the context of the Covid-19 outbreak since December 2019. These tests, based on the molecular recognition of the SARS-CoV-2 spike protein RBD (S-RBD) by specific highly potent antibodies, yield rapid, sensitive, and specific diagnosis. However, the SARS-CoV-2 spike protein is currently subject to many mutations.

Once mutated, the variants could lose their affinity towards the identified antibodies. However, the affinity between any variants of the SARS-CoV-2 spike protein RBD and the Angiotensin-Converting Enzyme 2 (ACE2) should be preserved. Indeed, the entry of the virus into the host cell involves interactions between ACE2 and S-RBD. [1][START_REF] Wrapp | Cryo-EM Structure of the 2019-NCoV Spike in the Prefusion Conformation[END_REF][START_REF] Yang | Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor[END_REF][START_REF] Li | Structure, function, and evolution of coronavirus spike proteins[END_REF] Consequently, ACE2 could be a target with limited mutation escaping possibilities. Some study report on the development of tools for the detection of the SARS-CoV-2 spike protein through its interactions with ACE2 in solution or on paper. [START_REF] Ahmad | Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current[END_REF] Nevertheless, as ACE2 has not evolved to recognize the spike protein, the affinity between ACE2 and S-RBD needs to be improved for clinical diagnosis use. [START_REF] Chan | Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2[END_REF] Several studies have investigated the interactions between ACE2 and the SARS-CoV-2 spike protein both to better understand the infection process, [START_REF] Piplani | In silico comparison of spike protein-ACE2 binding affinities across species; significance for the possible origin of the SARS-CoV-2 virus[END_REF][START_REF] Chen | Structure Analysis of the Receptor Binding of 2019-nCoV[END_REF][START_REF] Yan | Structural Basis for the Recognition of SARS-CoV-2 by Full-Length Human ACE2[END_REF][START_REF] He | Molecular Mechanism of Evolution and Human Infection with SARS-CoV-2[END_REF][START_REF] Sumbul | Dynamics and Binding Strength of the Spike Protein of Sars-Cov-2 Probed by High-Speed Atomic Force Microscopy[END_REF] and to develop therapeutic approaches. [START_REF] Chan | Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2[END_REF][START_REF] Hanson | Targeting ACE2-RBD Interaction as a Platform for COVID-19 Therapeutics: Development and Drug-Repurposing Screen of an AlphaLISA Proximity Assay[END_REF][START_REF] Basu | Molecular Docking Study of Potential Phytochemicals and Their Effects on the Complex of SARS-CoV2 Spike Protein and Human ACE2[END_REF][START_REF] Bernardi | Development and Simulation of Fully Glycosylated Molecular Models of ACE2-Fc Fusion Proteins and Their Interaction with the SARS-CoV-2 Spike Protein Binding Domain[END_REF] In particular, the crystal structure of ACE2 cellular receptor in complex with S-RBD has been resolved. [START_REF] Lan | Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor[END_REF][START_REF] Wang | Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2[END_REF] Wang et al. have identified 21 residues from S-RBD and 16 hydrogen bonds at the binding interface with ACE2, [START_REF] Wang | Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2[END_REF] while Lan et al. have reported 17 residues from S-RBD in contact with 20 residues of ACE2, allowing the formation of 13 hydrogen bonds and 2 salt bridges at the interface. [START_REF] Lan | Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor[END_REF] Also, by using a molecular docking approach, Chen et al.

have reported a stronger affinity between ACE2 and S-RBD than between ACE2 and the SARS-CoV-1 spike protein RBD, and identified a strong interaction between the residue F486 from S-RBD and the ACE2 hydrophobic pocket including the residues F28, L79, Y83 and L97. [START_REF] Chen | Structure Analysis of the Receptor Binding of 2019-nCoV[END_REF] Likewise, He et al. have determined by Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) the binding free energy between ACE2 and S-RBD. [START_REF] He | Molecular Mechanism of Evolution and Human Infection with SARS-CoV-2[END_REF] The complex ACE2 -S-RBD was found significantly more stable than the complex ACE2 -SARS-CoV-1 spike protein RBD. Moreover, Chan et al. have suggested thanks to deep mutagenesis and flow cytometry experiments that mutations in the asparagine 90-glycosylation motif of ACE2 could increase the affinity between ACE2 and S-RBD. Such ACE2 variant could be used to develop decoy receptors with high affinity. [START_REF] Chan | Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2[END_REF] From a therapeutic point of view, Yang et al. have combined Atomic Force Microscopy (AFM) experiments and Molecular Dynamics (MD) simulations, to investigate the inhibitor effect of ACE2-derived peptides to reduce the complex formation between ACE2 and S-RBD. [START_REF] Yang | Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor[END_REF] Protein adsorption is used in analytical devices as a simple immobilization mean to trap analytes to be detected through protein / analyte interactions. However, the orientation and conformation of the adsorbed proteins need to be controlled to preserve their ability to trap the targeted analyte. Among surfaces, silane monolayers are commercially available and widely used to immobilize proteins for biosensing purposes. However, it has been demonstrated that the nature of the silane monolayer influences the protein activity and trapping efficiency. [START_REF] Yang | Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer[END_REF][START_REF] Yang | Improvement of Protein Immobilization for the Elaboration of Tumor-Associated Antigen Microarrays: Application to the Sensitive and Specific Detection of Tumor Markers from Breast Cancer Sera[END_REF] For example, among three different aminated silane molecules, the ones terminated by Chitosan head-groups could allow the highest antigen-antibody binding capacity. [START_REF] Yang | Characterization of Three Amino-Functionalized Surfaces and Evaluation of Antibody Immobilization for the Multiplex Detection of Tumor Markers Involved in Colorectal Cancer[END_REF] Indeed, silane monolayers are expected to induce conformational changes in the adsorbed proteins which could lead to a weakening or a strengthening of protein -analytes interactions. [START_REF] Lecot | Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations[END_REF] MD simulations are well-adapted to investigate the conformation and orientation of protein adsorbed on surfaces, protein -surface interactions and protein -protein interactions at atomic scale. In a previous MD simulation study, we have investigated the adsorption of streptavidin on silane monolayers and the impact of streptavidin adsorption-induced conformational changes on its interactions with biotin. From the calculation of the streptavidin -biotin rupture force by steered molecular dynamic simulations (SMD), we have shown that silane molecules with charged head-groups, as well as silane molecules with long alkyl chains, induce conformational changes on streptavidin residues close to the biotin binding pocket which weaken streptavidin -biotin interactions. On the contrary, highly hydrophobic silane molecules with short alkyl chains and with neutral head-groups would lead to the lowest adsorptioninduced conformational changes in streptavidin, and to a streptavidin-biotin rupture force similar to that obtained, from SMD simulations and AFM experiments, [START_REF] Rico | Heterogeneous and Rate-Dependent Streptavidin-Biotin Unbinding Revealed by High-Speed Force Spectroscopy and Atomistic Simulations[END_REF] in the native state of the complex (i.e., in water without silane monolayer). [START_REF] Lecot | Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations[END_REF] In this work, we investigate by MD simulations, the effects of different silane monolayers on the adsorption of ACE2 (orientation of adsorbed ACE2, global and local adsorption-induced conformational changes), and the resulting effects on the affinity between ACE2 and S-RBD (conformational change in the residues at the interface between ACE2 and S-RBD, free binding energy and rupture force between adsorbed ACE2 and S-RBD). The six different silane monolayers investigated are elaborated from 3-aminopropyldimethylethoxysilane (C7H19NOSi named NH 3 + ), butyldimethylmethoxysilane (C7H18OSi named CH 3 short), octadecyldimethylmethoxysilane (C21H46OSi named CH 3 long), and tert-butyl-11-(dimethylamino(dimethyl)silyl)undecanoate (C19H41NO2Si, leading to COO -after deprotection), a mix (1:1) of CH 3 short and NH 3 + silane molecules (named mix short), and a mix (1:1) of CH 3 long and NH 3 + silane molecules (named mix long) (Table 1).

3-aminopropyldimethylethoxy-silane

Mix short

Mix CH 3 short: NH 3 + (1:1)

Mix long

Mix CH 3 long: NH 3 + (1:1)

Methods

This work is divided into three parts. Firstly, MD simulations of ACE2 on different types of silane monolayers were performed to select the monolayers that could lead to ACE2 adsorption with an orientation compatible with S-RBD trapping. Also, the adsorption-induced conformational changes in ACE2 were characterized. Secondly, three selected silane monolayers and the complex ACE2 -S-RBD were considered. Adsorption-induced conformational changes in ACE2 were characterized, especially at the interface with S-RBD and compared to the structure of the complex in water without surface. Finally, SMD simulations and Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) analysis gave insight into the impact of conformational changes on the strength of interactions between ACE2 and the S-RBD.

System description

MD simulations were performed from the PDB crystal structure 6M0J of the ACE2 -S-RBD complex. [START_REF] Lan | Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor[END_REF] Water molecules were kept while the zinc and chloride ions were not considered.

Regarding the surface, the amorphous SiO 2 layer was taken from Roscioni et al. [START_REF] Roscioni | Structural Characterization of Alkylsilane and Fluoroalkylsilane Self-Assembled Monolayers on SiO2 by Molecular Dynamics Simulations[END_REF] and resized to the simulation box dimensions with a 2nm-thickness by a home-made Python code. Silane monolayers were built following our process adapted from the method proposed by Roscioni et al. [START_REF] Roscioni | Structural Characterization of Alkylsilane and Fluoroalkylsilane Self-Assembled Monolayers on SiO2 by Molecular Dynamics Simulations[END_REF] , as we have previously described. [START_REF] Lecot | Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations[END_REF][START_REF] Lecot | Effects of the Chemical and Structural Properties of Silane Monolayers on the Organization of Water Molecules and Ions at Interfaces, from Molecular Dynamics Simulations[END_REF][START_REF] Lecot | Arrangement of Monofunctional Silane Molecules on Silica Surfaces: Influence of the Alkyl Chain Length, Head-Group Charge and Surface Coverage, from Molecular Dynamics Simulations, X-Ray Photoelectron Spectroscopy and Fourier Transform Infrared Spectroscopy Analysis[END_REF]

Simulations details

Simulations were achieved with the Gromacs software, version 5.1.3 [START_REF] Van Der Spoel | GROMACS: Fast, Flexible, and Free[END_REF] and visualization was performed with VMD, version 1.9.3. [START_REF] Humphrey | Visual Molecular Dynamics[END_REF] Energy minimization was done with the steepest descent method and the NVT and NPT equilibrations were performed for 100 ps each. Then, several production simulations were performed. The leap-frog algorithm was used for the integration of motion equations, with a time step of 2 fs. Constraints to bond parameters were applied with a LINCS algorithm. Temperature was kept at 300 K with a Nose-Hoover thermostat using a time constant of 0.4 ps, while pressure was maintained at 1 bar using a Parrinello-Rahman barostat and a time constant of 12 ps. The OPLS all-atom force-field [START_REF] Jorgensen | Development and Testing of the OPLS All-Atom Force Field on Conformational Energetics and Properties of Organic Liquids[END_REF] was used to describe ACE2 and S-RBD. The amorphous SiO 2 layer and silane molecules were described with a force-field adapted from previous studies [START_REF] Roscioni | Structural Characterization of Alkylsilane and Fluoroalkylsilane Self-Assembled Monolayers on SiO2 by Molecular Dynamics Simulations[END_REF][START_REF] Castillo | Characterization of Alkylsilane Self-Assembled Monolayers by Molecular Simulation[END_REF] and from the OPLS all-atom force-field, [START_REF] Jorgensen | Monte Carlo Simulations of the Hydration of Ammonium and Carboxylate Ions[END_REF] as we have previously reported. [START_REF] Lecot | Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations[END_REF][START_REF] Lecot | Arrangement of Monofunctional Silane Molecules on Silica Surfaces: Influence of the Alkyl Chain Length, Head-Group Charge and Surface Coverage, from Molecular Dynamics Simulations, X-Ray Photoelectron Spectroscopy and Fourier Transform Infrared Spectroscopy Analysis[END_REF] The OPLS all-atom force-field was also used to describe TIP4P water, [START_REF] Jorgensen | Comparison of Simple Potential Functions for Simulating Liquid Water[END_REF] Na + ions [START_REF] Aqvist | Ion-Water Interaction Potentials Derived from Free Energy Perturbation Simulations[END_REF] and Cl -ions, [START_REF] Chandrasekhar | Energy Component Analysis for Dilute Aqueous Solutions of Lithium (1+), Sodium (1+), Fluoride (1-), and Chloride (1-) Ions[END_REF] while parameters for the LJ wall were taken from a recent study. [START_REF] Kitabata | Molecular Dynamics Study on Wettability of Poly (vinylidene fluoride) Crystalline and Amorphous Surfaces[END_REF] Long-range electrostatic interactions were calculated with the Particle Mesh Ewald method and a cut-off of 1 nm. A cut-off distance of 1 nm was also used for LJ potentials.

Adsorption of ACE2 on six different silane monolayers. ACE2 adsorption process on the six different silane monolayers was investigated by defining 25 simulation systems. The first system, used as a reference, was ACE2 in water without silane monolayer. The 24 other systems contained an amorphous SiO 2 surface functionalized with one of the 6 silane monolayers (NH 3 + , CH 3 short, CH 3 long, COO -, mix short and mix long) and ACE2 initially set at 0.8 nm above the silane molecules with one of the four initial orientations defined in Figure 1. Initial orientations 2, 3 and 4 were obtained from the initial orientation 1, by a rotation of 180°, 90°

and -90° along the y axis, respectively. Simulation box dimensions were 10.6 nm × 10.6 nm × 19 nm. 3 independent simulation replicas of 100 ns were performed for each system. So, 75 simulations of 100 ns were performed in total. Images of some of these systems at final state are given in Figure 2. Adsorption of the ACE2 -S-RBD complex on the selected silane monolayers. The complex adsorption was investigated on the selected silane monolayers (i.e. the CH 3 short, mix short, and mix long monolayers) and from the initial orientations identified at the first step. The complex was initially positioned at 0.8 nm above the silane monolayers. Furthermore, another system was defined, including the complex in water without surface, which served as a reference. Simulation box dimensions were 13.8 × 13.8 × 24 nm. 6 independent simulation replicas of 200 ns were performed for each system. So, 24 simulations of 200 ns were performed in total. Representative images of the simulated systems at final state are given in Figure 3.

SMD simulations: S-RBD unbinding from ACE2. Initial configurations of Steered Molecular

Dynamics (SMD) simulations were selected among the final states of the 200-ns MD simulations modelling the complex in water and the complex adsorbed on the silane monolayers mix short and mix long. During SMD simulations, an external force was applied on the centerof-mass of S-RBD to pull it away from ACE2. Regarding the system with the complex in water, a position restriction was applied on the ACE2 center-of-mass to prevent its movement and to allow the detachment of S-RBD (the position restriction was applied on the ACE2 atom closest to its center of mass, excepting hydrogen atoms). On the contrary, no position restriction was applied on ACE2 for the systems where it was adsorbed on a silane monolayer, and no covalent bond was defined with the surface. Indeed, the Van der Waals and electrostatic interactions which were established between ACE2 and the SiO 2 layer functionalized with silane molecules were strong enough to prevent its desorption while S-RBD was pulled away by the applied force. This force was a harmonic potential, with a force constant k = 100 pN/nm and a pulling velocity v = 2 m.s -1 . To reproduce AFM experiments, the force was applied on the spike protein in the direction normal to the surface. Two initial configurations were considered for each system. Simulation boxes were extended up to 32 nm to verify the minimum image convention.

Energy minimization, NPT equilibration and simulation productions of 6 ns were performed.

20 independent simulation replicas were achieved for each starting configuration.

Analysis parameters

The following parameters were investigated by using Gromacs analysis functions, g_mmpbsa tools, VMD tools and home-made Python codes. Error bars correspond to the standard error of the values calculated for the different simulation replicas.

The interaction energies between the silane monolayers and the ACE2 -S-RBD complex were investigated through the Van der Waals and electrostatic contributions. The ACE2 adsorption-induced conformational changes were investigated by calculating the root-mean-square deviation (RMSD) and the root-mean-square fluctuation (RMSF), and the variations in the protein secondary structure. Also, the number of hydrogen bonds which are established between ACE2 and S-RBD were calculated. Finally, the contact surface between two elements is defined as following: atoms from ACE2 are considered in contact with the silane monolayer or with S-RBD if they are located at less than 0.4 nm from one atom of a silane molecule or of S-RBD, respectively. The given values are averaged over the last 10 ns of the MD simulations (100 ns or 200 ns).

MMPBSA analysis.

Binding free energy between ACE2 and S-RBD (ΔG) is determined by MMPBSA with the g_mmpbsa tool. [START_REF] Kumari | g_mmpbsa -A GROMACS Tool for High-Throughput MM-PBSA Calculations[END_REF] Due to the approximations and hypothesis made during the calculation, MMPBSA results should not be interpreted quantitatively, but should be compared qualitatively between the different systems considered. [START_REF] Ali | Dynamics of the ACE2-SARS-CoV-2/SARS-CoV spike protein interface reveal unique mechanisms[END_REF] The given values are calculated over the last ns of the simulation (one point every 100 ps).

SMD simulations. The rupture force was calculated from the force -time curves and defined as the highest force applied on S-RBD before its detachment. Rupture force and detachment time were determined for each simulation replica. Mean values, standard deviation and histograms of the rupture force were calculated.

Results and Discussion

Identification of the silane monolayers which are the most appropriate to adsorb the ACE2 cellular receptor with an orientation compatible with the trapping of S-RBD.

For biosensing purposes, we firstly deciphered if some silane monolayers could allow the systematic adsorption of ACE2, with an orientation compatible with the trapping of S-RBD.

Because of computational limitations, as simulations cannot be long enough to allow global reorientation of ACE2 around the silane monolayers before adsorption, four initial orientations of ACE2 are studied, as shown Figure 1.

Adsorption did barely influence the initial ACE2 orientations. The Van der Waals (VdW) and the electrostatic (elec) contributions to the interaction energy between ACE2 and the silane monolayers are calculated over three replicates, for each initial orientation. For each silane monolayer we defined the most likely trapping orientation as the one that leads both to a systematic adsorption of ACE2 (through all replicates) and to the most favorable interaction energy. Among the four orientations studied, orientation 1 is the best suited as the residues interacting with S-RBD are located on the top of the protein, exposed to the water bulk. On the contrary, ACE2 cannot interact with S-RBD with orientation 2, as interfacial residues are then located below the protein, in contact with the silane molecules. Orientations 3 and 4 seem to allow interactions with S-RBD but might be less favourable than orientation 1. Indeed, in the case of biosensing applications with several ACE2 adsorbed on the surface, orientations 3 and 4 could lead to steric hindrances that might disturb S-RBD trapping. Evolution of the total VdW and elec interaction energy between ACE2 and the surface is shown in Supporting information, Figure S1, while immobilization frequencies (over three replicates) and interactions energies after ACE2 adsorption on silane monolayers are summarized in Supporting Information, Table S1.

on the surface only for one of the three simulation replicates, and after 45 ns. With orientations 2, 3 and 4, the ACE2 cellular receptor is rapidly adsorbed on the surface for all simulation replicas (less than 15 ns). The interaction energy is progressively stabilized, and initial orientation 4 leads to the most favourable adsorption (i.e. to the lowest VdW and elec interaction energies, about -122 kcal/mol). For NH 3 + monolayer, ACE2 cellular receptor is quickly and strongly adsorbed on the surface for all simulation replicas, regardless of its initial orientation (interaction energy varies between -258 kcal/mol and -386 kcal/mol depending on the initial orientation). However, the most likely orientation seems to be orientation 2 (-386 kcal/mol), which is not suitable for our application. Concerning the CH 3 long monolayer, adsorption on silane monolayer for all simulation replicas is only observed with orientation 3.

However, the immobilization process is long, as interactions with the silane molecules appear after more than 30 ns, 70 ns and 20 ns for each simulation replicas. In line with the long adsorption process, the interaction energy between ACE2 and the CH 3 long monolayer is weak, about -61 kcal/mol. Thus, the CH 3 long monolayer does not seem to be suitable for ACE2 adsorption. Regarding the COO -monolayer, few or no interaction appears between ACE2 and the surface with ACE2 initial orientations 2, 3, and 4, while interactions appear after several tens of ns in the case of orientation 1. However, a positive interaction energy between ACE2 and COO -silane molecules is obtained with orientation 1, showing that these interactions are not favourable. This could be explained by the non-favourable electrostatic interactions occurring between ACE2 (that is negatively charged -27 e) and the negative head-group charge of the silane molecules. Concerning the mix short monolayer, ACE2 cellular receptor is rapidly immobilized on the surface for all initial orientations (less than 5 ns, Supporting Information, Figure S1). Furthermore, orientation 1 is the most likely regarding the interaction energy (about -245 kcal/mol), while orientation 2 is the less likely (about -172 kcal/mol). Eventually, ACE2 cellular receptor is adsorbed on the mix long silane monolayer for all initial orientations and simulation replicas, orientation 1 being the most likely (about -256 kcal/mol) and orientation 2 the less likely (about -201 kcal/mol). Thus, the mix short and the mix long silane monolayers seem well-suited to immobilize ACE2 with similar interaction energies (around -250 kcal/mol) with an orientation compatible with the trapping of S-RBD. Indeed, on these two monolayers the most probable ACE2 trapping orientation is the orientation 1. The CH 3 short silane monolayer could also be suitable for immobilizing ACE2, even if less favourable than the mix short and mix long monolayers. Indeed, the most likely ACE2 adsorption orientation is the orientation 4 (interaction energy around -122 kcal/mol), that could lead to a steric hindrance for S-RBD trapping if several ACE2 were adsorbed on the monolayer (Figure 2).

Conformational changes induced in ACE2 by its adsorption on the CH 3 short, mix short, and mix long silane monolayers are characterised by calculating the Root Mean Square Deviation (RMSD) of ACE2 (Supporting Information, Figure S2). RMSD values are 3.2 ± 0.3 Å in water, 3.0 ± 0.1 Å when ACE2 is immobilized on CH 3 short monolayer, 3.4 ± 0.1 Å on mix short monolayer and 3.1 ± 0.2 Å on mix long monolayer (these values are calculated from the initial simulation step and the last 10 ns of the simulations, i.e. when the system is stabilized). RMSD values when ACE2 is adsorbed on silane monolayers are close to the one obtained in water.

Thus, as for the streptavidin -biotin complex, [START_REF] Lecot | Impact of Silane Monolayers on the Adsorption of Streptavidin on Silica and its Subsequent Interactions with Biotin: Molecular Dynamics and Steered Molecular Dynamics Simulations[END_REF] we suggest that the conformational changes induced by adsorption on silane monolayers are quite low, allowing to preserve the global structure of ACE2. Furthermore, Root Mean Square Fluctuations (RMSF) traces show that the mobility of ACE2 residues interacting with S-RBD is quite similar to the one observed in water (Supporting Information, Figure S3). Thus, it seems that the ability of ACE2 to interact with S-RBD could be preserved after its adsorption on the three identified silane monolayers.

In the following, only the three identified silane monolayers (mix short, mix long, and CH 3 short) are studied.

Adsorption induced conformational changes in the ACE2 -S-RBD complex.

Interactions between the ACE2 -S-RBD complex and the CH 3 short, mix long and mix short silane monolayers. It has been previously demonstrated that the adsorption and orientation of protein on charged surfaces is controlled by its electric dipole. [START_REF] Quan | Multiscale modeling and simulations of protein adsorption: progresses and perspectives[END_REF][START_REF] Zhou | Molecular simulation studies of the orientation and conformation of cytochrome c adsorbed on self-assembled monolayers[END_REF] Thus, we have plotted the electric dipole moment [START_REF] Jurrus | Improvements to the APBS biomolecular solvation software suite[END_REF] of the ACE2-S-RBD complex adsorbed on the mix short and mix long monolayers in Supporting Information, Figure S4. The dipole moment orientations are well coherent with the complex adsorption on a positively charged surface.

Interactions between the complex and the silane monolayers CH3 short, mix short and mix long were characterized by the interaction energy, the number of residues in the contact area between ACE2 and silane molecules and the type of ACE2 residues which are likely to interact with silane molecules (Table 2).

Table 2. Total VdW and elec interaction energy between the ACE2 -S-RBD complex and the silane monolayers, number of residues in the contact area between ACE2 and the silane monolayers, number of residues in the contact area between S-RBD and the silane monolayers, and list of ACE2 residues in contact with the silane monolayers. The interaction energy between the complex and silane molecules are lower with the mix long monolayer than with the mix short and CH 3 short silane monolayers while the contact area are higher. Indeed, with the mix long monolayer, numerous Van der Waals interactions are established between the long alkyl chains of CH 3 long silane molecules and ACE2 hydrophobic residues (A, L, P, V, I, M, F, W), in addition to electrostatic interactions between the NH 3 + silane molecules and polar or negatively charged ACE2 residues (D, E).

System

Mix short silane monolayer shows a lower interaction energy and a lower contact surface with ACE2 than the CH 3 short silane monolayer. Indeed, electrostatic interactions are established between the positively charged NH 3 + silane molecules of the mix short monolayer and ACE2 negatively charged residues (D609, D615). However, NH 3 + silane molecules have also a repulsive impact on ACE2 positively charged residues, which could explain the lower contact surface. Furthermore, due to ACE2 orientation on the CH 3 short monolayer, some interactions are established between S-RBD and the silane molecules. Finally, the mix long and mix short monolayers seem to have the highest affinities with the studied complex. In the following, we characterize the structure of the complex adsorbed on the CH 3 short, mix short, and mix long monolayers.

RMSD and secondary structure. RMSD values of the ACE2 -S-RBD complex after ACE2 adsorption on the three studied silane monolayers are summarized in Table 3, while its secondary structure is characterized in Supporting Information, Table S2. The RMSD values of ACE2, of S-RBD, and of the complex in water are below 4.5 Å, in agreement with the value determined by Ali et al. from MD simulations. [START_REF] Ali | Dynamics of the ACE2-SARS-CoV-2/SARS-CoV spike protein interface reveal unique mechanisms[END_REF] So, the complex structure integrity, in water, is preserved through the simulation. Furthermore, the structure of the complex adsorbed on the studied silane monolayers seems well preserved, as its RMSD values are close to the one obtained in water. Also, the complex secondary structure is wellpreserved after its adsorption on each studied silane monolayers (Table S2). So, these results suggest, once again, that the affinity between ACE2 adsorbed on the studied silane monolayers and S-RBD could be preserved.

RMSF.

To further characterize adsorption-induced conformational changes in the structure of the ACE2 -S-RBD complex, RMSF traces of the C α backbone of ACE2 and of S-RBD are given in Figure 4 for all systems, and summarized in Figure 5. In the case of the complex in water, the residues with high mobility are in accordance with the results from Ali et al. [START_REF] Ali | Dynamics of the ACE2-SARS-CoV-2/SARS-CoV spike protein interface reveal unique mechanisms[END_REF] Furthermore, the global evolution of RMSF traces is globally conserved when ACE2 is adsorbed on silane monolayers, in accordance with the above RMSD and secondary structure results. However, a few differences are observed for the ACE2 residues which are localized around the contact area with the silane monolayers, and around the interface with S-RBD. 280 to 300 and around the residue 427 than for the complex in water. These residues are close to the centre of ACE2. Regarding ACE2 residues which belong to the interface with S-RBD, a short decrease in the RMSF values is observed near the residues 24 to 45, 79 to 83, and 353 to 357 for the three silane monolayers. However, the decrease is greater in the case of the CH 3 short monolayer, likely because of additional interactions between S-RBD and the monolayer.

Regarding S-RBD, RMSF traces are slightly modified when ACE2 is adsorbed on the silane monolayers, compared to the values obtained for the complex in water. Regarding the CH 3 short monolayer, a higher mobility is observed around the residues 367 to 372, which are in contact with some silane molecules in several simulation replicas. For all silane monolayers, some variations in the RMSF values are observed around the residues 472 to 476, and 484 to 505, which belong to the interface with ACE2. These results suggest that some small conformational changes appear in the complex when ACE2 is adsorbed on the CH 3 short, mix short and mix long silane monolayers, especially around the residues interacting with the silane molecules, but also for the residues located at the ACE2 / S-RBD interface. These changes are higher in the case of the CH 3 short monolayer than in the case of the mix short and mix long monolayers, likely because of the additional interactions between S-RBD and the CH 3 short molecules. This could induce some modifications in the nature of the residues belonging to the interface between ACE2 and S-RBD.

Interface between ACE2 and S-RBD. The residues located at the interface between ACE2

and S-RBD are summarized in Figure 5 and defined as the ACE2 or S-RBD residues having one atom located at less than 0.4 nm from an atom from the other protein.

Results show that 19 ACE2 residues belong to the interface for the complex in water and when it is adsorbed on the mix long silane monolayer, while 20 residues belong to the interface with the CH 3 short monolayer, and 21 with the mix short monolayer. The five ACE2 residues identified by Laurini et al as hot spots, [START_REF] Laurini | Computational Alanine Scanning and Structural Analysis of the SARS-CoV-2 Spike Protein/Angiotensin-Converting Enzyme 2 Complex[END_REF] i.e., the main interactions sites with S-RBD are D38, K31, E37, K353 and Y41. These residues belong to the interface of all the studied systems, as well as eleven additional residues Q24, T27, F28, D30, H34, E35, Q42, L45, L79, M82 and Y83, in accordance with previous studies. [START_REF] Lan | Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor[END_REF][START_REF] Wang | Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2[END_REF] Furthermore, the residue I21 does not belong to the interface for the complex in water, but it does with the three silane monolayers. Also, some disparities appear depending on the type of silane monolayers. In particular, the residue R393 belongs to the interface in the case of the mix short monolayer, while for the mix long and CH 3 short monolayers, the residue Q76 belongs to the interface, which is perturbed around the residues G354 to R357, in accordance with the RMSF values.

Regarding S-RBD residues, 22 of them belong to the interface with ACE2 when the complex is in water, while 23 residues belong to the interface when ACE2 is adsorbed on the CH 3 short and mix short monolayer, and 24 with the mix long monolayer. Regarding the three hot-spot residues identified by Laurini et al., [START_REF] Laurini | Computational Alanine Scanning and Structural Analysis of the SARS-CoV-2 Spike Protein/Angiotensin-Converting Enzyme 2 Complex[END_REF] Q498 and T500 residues are still located at the interface with ACE2 when it is adsorbed on the silane monolayers, but not R403. These results are in agreement with the RMSF calculations. A few other modifications are observed. Especially, the L492 residue is added to the interface for all silane monolayers.

These results suggest that the interface between ACE2 and S-RBD is impacted by ACE2 adsorption-induced conformational changes, which depend on the type of silane monolayer.

This result could explain the increase in the number of hydrogen bonds between ACE2 and S-RBD observed when ACE2 is adsorbed on silane monolayers (Table 3). So, our results suggest that the interactions between ACE2 and S-RBD could be reinforced through ACE2 adsorptioninduced conformational changes.

Effect of silane monolayers on the binding free energy and rupture force between adsorbed ACE2 and S-RBD.

MMPBSA analysis. Binding free energy between ACE2 and S-RBD were determined from MMPBSA calculations (Table 4) for the complex in water without surface and for ACE2 adsorbed on the CH 3 short, mix short and mix long monolayers. Due to the calculation method approximations, results are not interpreted quantitatively, but qualitatively compared between the different systems. Results show that the adsorption of ACE2 on silane monolayers lead to a decrease in the binding free energy, in comparison with the complex in water, suggesting a stabilization of the interactions between ACE2 and S-RBD. These results can be partly explained by the increase in the number of hydrogen bonds observed between ACE2 and S-RBD (Table 3). To further understand the stabilization of the interactions between the adsorbed ACE2 and S-RBD, the contribution of each residue to the binding free energy was investigated. The binding energy change on each residue, i, is defined by analogy with the study of Laurini et al. [START_REF] Laurini | Computational Alanine Scanning and Structural Analysis of the SARS-CoV-2 Spike Protein/Angiotensin-Converting Enzyme 2 Complex[END_REF][START_REF] Laurini | Computational Mutagenesis at the SARS-CoV-2 Spike Protein/Angiotensin-Converting Enzyme 2 Binding Interface: Comparison with Experimental Evidence[END_REF] :

(1) ∆∆𝐺 𝑖 = ∆𝐺 𝑖 𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑖𝑛 𝑤𝑎𝑡𝑒𝑟 -∆𝐺 𝑖 𝑐𝑜𝑚𝑝𝑙𝑒𝑥 𝑎𝑑𝑠𝑜𝑟𝑏𝑒𝑑 𝑜𝑛 𝑠𝑖𝑙𝑎𝑛𝑒 𝑚𝑜𝑛𝑜𝑙𝑎𝑦𝑒𝑟 Thus, if , the residue contribution is stabilizing the ACE2 -S-RBD interactions, while ∆∆𝐺 𝑖 ≥ 0 if , the residue contribution shows a destabilizing effect. The residues with ∆∆𝐺 ≤ 0 | kcal/mol, i.e., the main residues contributing to the stabilization or to the ∆∆𝐺 | ≥ 0.4 destabilization of the complex are shown in Figure 6 and summarized in Figure 5, for each different silane monolayers. Most of the residues presented in Figure 6, i.e. the main residues contributing to the stabilization or to the destabilization of the adsorbed complex, are located at the interface between ACE2 and S-RBD (Figure 6). In particular, the apolar residue M82 from ACE2 and the polar or charged residues E484 and Q493 from S-RBD show a stabilizing effect for all silane monolayers. However, among the all residues presented Figure 6, those identified as interfacial hot-spot residues by Laurini et al. [START_REF] Laurini | Computational Alanine Scanning and Structural Analysis of the SARS-CoV-2 Spike Protein/Angiotensin-Converting Enzyme 2 Complex[END_REF] have too large error bars to show a strong and clearly stabilizing or destabilizing effect when ACE2 is adsorbed on the studied monolayers, except K31 that has a clearly stabilizing effect in the case of the CH 3 short monolayer (Supporting Information, page S7). Furthermore, some polar or charged residues which do not belong to the interfacial residues identified in Figure 6 show a stabilizing effect, whatever the studied silane monolayer, such as the residue E23 from ACE2 and the residue K458 from S-RBD. Also, the contribution of polar and charged residues in the stabilization of the complex is higher in the case of the CH 3 short monolayer (8.9 kcal/mol) than in the case of the mix short (7.6 kcal/mol) and mix long (7.5 kcal/mol) monolayers. This result could explain the highest complex stabilization observed with the CH 3 short monolayer. So, the increase in the binding free energy, induced by the adsorption of ACE2 on the three studied silane monolayers, can be largely explained both by the formation of novel hydrogen bonds and by the increase in the number of electrostatic interactions between particularly mobile residues of ACE2 and of S-RBD.

Finally, we notice that the sum of the stabilizing and destabilizing contributions of the residues presented in Figure 6 explains most of the global stabilization of the complex (Table 4). Indeed, in the case of the mix short monolayer, mix long, and CH 3 short monolayers, the 24, 16, and 24 residues with kcal/mol induce a total stabilization of 10.5 kcal/mol out of 11.7 | ∆∆𝐺 | ≥ 0.4 kcal/mol, 8.9 kcal/mol out of 9.9 kcal/mol, and 9.9 kcal/mol out of 15.2 kcal/mol. SMD simulations. The increase in the free binding energy between ACE2 and the SARS-CoV-2 spike protein due to ACE2 adsorption on the studied silane monolayers was further investigated by SMD simulations. SMD simulations are performed by applying a harmonic potential to the center-of-mass of S-RBD in the direction normal to the surface. The system with the CH 3 short monolayer is not suitable for SMD simulations since S-RBD interacts with the silane molecules. Thus, SMD simulations are only performed on the ACE2 -S-RBD complex in water without surface and on the complex adsorbed on the mix short and mix long silane monolayers.

For each system, SMD simulations are run from two different initial configurations, selected among the final states of the simulation replicas modelling the complex in water and the complex adsorbed on the mix short and mix long monolayers. In this way, the effect of the variability in the final orientation of the adsorbed complex (highlighted through the different simulation replicas for each silane monolayer) are taken into account. For each initial configuration, 20 SMD simulation replicas are performed with a pulling velocity v = 2 m.s -1 and a force constant k = 100 pN/nm. Force time curves are shown in Figure 7, while the mean rupture force values are summarized in Supporting information, Table S3 and histograms of the rupture forces are given in Supporting information, Figure S5. Regarding the ACE2 -S-RBD complex in water without surface and when the complex is adsorbed on the mix short monolayer, the mean rupture forces from the two initial configurations are close, while with the mix long silane monolayer, we observe different rupture forces for the two initial configurations. These results are in line with the error bars observed in the binding free energy calculations and may be explained by the variability in the adsorption-induced conformational changes for each system. The higher variability obtained with the mix long monolayer could be explained by the lower interactions between ACE2 and S-RBD. However, in line with the binding free energy calculations a clear effect of silane monolayers seems to appear. Indeed, adsorption on the mix short monolayer would lead to a rupture force higher (758 pN and 715 pN for initial configurations 1 and 2) than in water without surface (607 pN and 623 pN for initial configurations 1 and 2). Regarding the mix long monolayer, the rupture force obtained with one of the initial configurations is higher than the values obtained for the complex in water (743 pN), while the value obtained with the other configuration is slightly lower (537 pN). However, results from MMPBSA analysis, which consider the six simulation replicas, would indicate a global increase in the binding free energy between ACE2 and S-RBD when ACE2 is adsorbed on the mix long silane monolayer. So, as shown both from MMPBSA calculations and SMD simulations, the studied silane monolayers would tend to increase by 20% the affinity between ACE2 and S-RBD through the formation of novel hydrogen bonds and electrostatic interactions at the interface between ACE2 and S-RBD.

Conclusions

The adsorption of ACE2 on six different silane monolayers was studied by MD simulations.

Among them, it was demonstrated that three silane monolayers, CH 3 short, mix short and mix long would allow to adsorb ACE2 with an orientation compatible with S-RBD trapping. Indeed, from considerations on the Van der Waals and electrostatic interaction energies, S-RBD binding pocket on ACE2 should be more likely exposed to the bulk water, in the case of ACE2 adsorption on the mix short and mix long monolayers. In the case of ACE2 adsorption on the CH 3 short monolayer, S-RBD binding pocket on ACE2 should be close to the silane molecules but still accessible. RMSD and RMSF, which give information on the global and per residues conformational changes, appeared similar for ACE2 in water without surface and for ACE2 adsorbed on the studied silane monolayers. So, adsorption on the silane monolayers should not impact the ability of ACE2 to trap S-RBD. The adsorption of the ACE2 -S-RBD complex leads to slight conformational changes in residues around the interface between ACE2 and S-RBD. Our results suggest that these adsorption-induced conformational changes lead to the formation of novel hydrogen bonds and electrostatic interactions which increase the native affinity between ACE2 and S-RBD. Interactions are further increased with the CH 3 short monolayer, than with the mix short monolayer and, than with the mix long monolayer. These results are in line with the binding free energy calculated by MMPBSA and the rupture forces calculated by SMD simulations between ACE2 and S-RBD. Consequently, from considerations on the binding free energy but also on the most likely orientation of adsorbed ACE2, the mix short monolayer might be well-suited to adsorb ACE2, while increasing by 20% its native affinity towards the SARS-CoV-2 spike protein for biosensing applications. The mix long monolayer could also be suitable, despite more variability in the results likely because of the smaller increase in the binding free energy. Such a 20% increase in the binding free energy could help to improve the efficiency of the current tools using ACE2 for S-RBD detection. [START_REF] Ahmad | Development of an Optical Assay to Detect SARS-CoV-2 Spike Protein Binding Interactions with ACE2 and Disruption of these Interactions Using Electric Current[END_REF] Also, this study suggests that by combining ACE2 adsorption on specific silane monolayers and the use of ACE2 variants, [START_REF] Chan | Engineering human ACE2 to optimize binding to the spike protein of SARS coronavirus 2[END_REF] biosensing tools with efficiency that competes with antigenic tests, and towards any variants of the SARS-CoV-2 spike protein, could be developed. In order to sustain our MD results on the adsorption of ACE2 on silane monolayers, experimental evidences would be needed. Although, the in-situ investigations of proteins at solid/liquid interface remain challenging, a combination of different techniques could help to gain experimental insights. Among them one can envisioned to study the structure of immobilized ACE2 by AFM in the Single Molecule Force Spectroscopy mode, [START_REF] Yang | Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor[END_REF] by Sum Frequency Generation vibrational spectroscopy, [START_REF] Guo | Molecular Structure of the Surface-Immobilized Super Uranyl Binding[END_REF][START_REF] Hosseinpour | Structure and Dynamics of Interfacial Peptides and Proteins from Vibrational Sum-Frequency Generation Spectroscopy[END_REF] and to measure the affinity between ACE2 and monoclonal antibodies or/and SARS-CoV-2 spike protein RBD using Surface Plasmon Resonance. On a longer time scale perspectives, the experimental validation of MD simulations could help us to select a-priori specific surface modification for protein immobilisation and then fasten the development of biosensor interfaces.

SUPPORTING INFORMATION

The following files are available free of charge.

Total VdW and elec interaction energy between the different silane monolayers and ACE2 cellular receptor. RMSD of ACE2 cellular receptor. RMSF traces of ACE2 cellular receptor.

Figure 1 .

 1 Figure 1. Illustration of the four initial orientations of the ACE2 cellular receptor above the

Figure 2 .

 2 Figure 2. Representative images of four different systems at final state (after 100 ns MD

Figure 3 .

 3 Figure 3. Representative images of the four different systems at final state (after 200 ns MD

Figure 4 .

 4 Figure 4. RMSF traces of the C α atoms of ACE2 adsorbed on silane monolayers (a) CH 3

Figure 5 .

 5 Figure 5. ACE2 and S-RBD residues with a mobility different from that in water without

Figure 6 .

 6 Figure 6. Binding free energy change ( obtained from MMPBSA calculations for the ∆∆𝐺 𝑖 )

Figure 7 .

 7 Figure 7. Typical force-time curves determined by SMD simulations for the ACE2 -S-RBD

19

Table 1 .

 1 Composition of the six silane monolayers.

	Silane monolayer	Silane molecule	Silane structure
	CH 3 short	butyldimethyl-methoxysilane
	CH 3 long	octadecyl-dimethyl-methoxysilane
		(dimethylamino)-
	COO -	dimethylsilylun-
		decanoate

Table 3 .

 3 RMSD values and number of hydrogen bonds between S-RBD and ACE2 for all systems studied.

	System	RMSD complex (Å)	RMSD ACE2 (Å)	RMSD S-RBD (Å)	Number of hydrogen bonds
	In water	4.1 ± 0.2	3.9 ± 0.2	4.5 ± 0.4	9.1 ± 0.6
	CH 3 short	4.2 ± 0.4	3.8 ± 0.2	5.3 ± 0.7	10.3 ± 0.7
	Mix short	3.7 ± 0.2	3.5 ± 0.2	4.1 ± 0.2	11.9 ± 0.7
	Mix long	4.2 ± 0.2	3.9 ± 0.2	4.8 ± 0.2	10.5 ± 0.6

Table 4 .

 4 Binding free energy between ACE2 and S-RBD calculated by MMPBSA.

		System	In water	CH 3 short	Mix short	Mix long
	∆𝑮	(kcal/mol)	-63.0 ± 2.6	-78.2 ± 2.7	-74.7 ± 3.7	-72.9 ± 4.0

Funding Sources

This work was supported by the Young Researcher ANR PORIDG project, grant ANR-18-CE09-0006 of the French Agence Nationale de la Recherche. This work was granted access to the HPC resources of CINES under the allocation 2019-A0070711100 made by GENCI. Also, this work was supported by the PMCS2I -supercomputer Newton from Ecole Centrale de Lyon, France, member of the FLMSN.

Secondary structure of the ACE2 -S-RBD complex. MMPBSA analysis. Unbinding time and rupture force from SMD simulations. Rupture force distribution from SMD simulations. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Email: christelle.yeromonahos@ec-lyon.fr

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

ACKNOWLEDGMENT

The authors thank Laurent Pouilloux, Anne Cadiou, and Laurent Carrel for support on PMCS2I resources. S. L. is grateful to the "doctoral school Material" from the University of Lyon, France, for offering a Ph.D. fellowship.