key: cord-329840-f3dsu36p
authors: Hati, Sanchita; Bhattacharyya, Sudeep
title: Impact of Thiol-Disulfide Balance on the Binding of Covid-19 Spike Protein with Angiotensin Converting Enzyme 2 Receptor
date: 2020-05-11
journal: bioRxiv
DOI: 10.1101/2020.05.07.083147
sha: 
doc_id: 329840
cord_uid: f3dsu36p

The novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to an ongoing pandemic of coronavirus disease (COVID-19), which started in 2019. This is a member of Coronaviridae family in the genus Betacoronavirus, which also includes SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). The angiotensin-converting enzyme 2 (ACE2) is the functional receptor for SARS-CoV and SARS-CoV-2 to enter the host cells. In particular, the interaction of viral spike proteins with ACE2 is a critical step in the viral replication cycle. The receptor binding domain of the viral spike proteins and ACE2 have several cysteine residues. In this study, the role of thiol-disulfide balance on the interactions between SARS-CoV/CoV-2 spike proteins and ACE2 was investigated using molecular dynamic simulations. The study revealed that the binding affinity was significantly impaired when all the disulfide bonds of both ACE2 and SARS-CoV/CoV-2 spike proteins were reduced to thiol groups. The impact on the binding affinity was less severe when the disulfide bridges of only one of the binding partners were reduced to thiols. This computational finding provides a molecular basis for the severity of COVID-19 infection due to the oxidative stress.

The novel coronavirus known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or simply COVID-19 is the seventh member of the coronavirus family. 1 The other two viruses in this family that infect humans are severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). These are positive-sense, single-strand enveloped RNA viruses. The coronavirus particles contain four main structural proteins: the spike, membrane, envelope, and nucleocapsid. [1] [2] The spike protein protrudes from the envelope of the virion and consists of two subunits; a receptor-binding domain (RBD) that interacts with the receptor proteins of host cells and a second subunit that facilitates fusion of the viral membrane into the host cell membrane. Recent studies showed that RBD of spike proteins of SARS-CoV and SARS-CoV-2 interact with angiotensin-converting enzyme 2 (ACE2). ACE2 belongs to the membrane-bound carboxydipeptidase family. It is attached to the outer surfaces of cells and is widely distributed in the human body. In particular, higher expression of ACE2 is observed in organs such as small intestine, colon, kidney, and heart, while ACE2 expression is comparatively lower in liver and lungs. [3] [4] The role of oxidative stress on the binding of viral proteins on the host cell surface receptors is a relatively underexplored area of biomedical research. [5] [6] [7] [8] [9] [10] Previous studies have indicated that the entry of viral glycoprotein is impacted by thiol-disulfide balance on the cell surface. 5, [7] [8] [9] [11] [12] Any perturbations in the thiol-disulfide equilibrium has also been found to deter the entry of viruses into their target cells. 5 The first step of the viral entry involves binding of the viral envelop protein onto a cellular receptor. This is followed by endocytosis, after which conformational changes of the viral protein helps the induction of the viral protein into the endosomal membrane, finally releasing the viral content into the cell. These conformational changes are mediated by pH changes as well as the conversion of disulfide to thiol group of the viral spike protein. 7 Several cell surface oxidoreductases 9 regulate the thiol-disulfide exchange, responsible for conformational changes of viral proteins needed for virus entry into host cells.

In the backdrop of significant mortality rate for SARS-CoV-2 (hereinafter referred to as CoV-2) infection, it is important to know if the thiol-disulfide balance plays any role on the binding of the spike glycoprotein on to the host cell receptor protein ACE2. A recent study with the spike glycoprotein of SARS-CoV (hereinafter referred to as CoV) has exhibited a complete redox insensitivity; 7 despite the reduction of all disulfide bridges of CoV to thiols, its binding to ACE2 remained unchanged. 7 However, this study did not probe the redox sensitivity of ACE2 receptor. Thus, in the present study, we computationally investigated the redox state of both partners (ACE2 and CoV/CoV-2) on their binding affinities. The structure of CoV 1 3 and CoV-2 14-15 complexed with ACE2 are known and the noncovalent interactions at the protein-protein interface 16 have been reported recently. Using these reported structures, molecular dynamics simulations and electrostatic field calculations were performed to explore the impact of thioldisulfide balance on CoV/CoV-2 and ACE2 binding affinities. The structural and dynamical changes due to the change in the redox states of cysteines in the interacting proteins were analyzed and their effects on binding free energies were studied.

The molecular basis of the binding of spike proteins to ACE2 is known from X-ray crystallographic (SARS-CoV) 13 and cryo-electron microscopic (SARS-CoV-2) 16 studies. The sequence alignment of CoV and CoV-2 spike proteins showed high sequence identity (>75 %)

indicating that their binding to ACE2 receptors will be similar ( Fig. 1 ). In both bound structures, the RBD of CoV and CoV-2 is found to be complexed with ACE2 (Fig. 2) . Both ACE2 and

CoV-2 possess four disulfide bridges, whereas CoV subunit has only two disulfide linkages (Table 1 and Fig. 2a and 2b) . Two large helices of ACE2 form a curved surface (Fig. 2, illustrated by the dashed curved line) that interacts with the concave region of CoV or CoV-2 subunit.

Structural change along the trajectory. In all cases, the simulation started with an equilibrated structure, which was obtained after minimizing the neutralized solvated protein complex built from the experimentally determined structures. The evolution of the protein structure along the MD trajectory was monitored by calculating the root-mean-square deviation (RMSD) of each structure from the starting structure as a frame of reference following standard procedure. 17 Briefly, during the MD simulation, the protein coordinates were recorded for every 10 ps interval and a root-mean-square-deviation (RMSD) of each frame was calculated from the average root-mean-square displacement of backbone C α atoms with respect to the initial structure. Then, the RMSD values, averaged over conformations stored during 1 ns time, were plotted against the simulated time (Fig. 3 ). Compared to the starting structure, only a moderate backbone fluctuation was noted in all protein complexes during 20 ns simulations and the maximum of RMSD was in the range of 2.0-3.8 Å ( Table 2 ). The evolution was smooth and its stability was demonstrated by the standard deviation of the computed RMSDs, which was less than 0.3 Å. Taken together, results of the simulations showed no unexpected structural deformation of the SAR-CoV/CoV-2⋅⋅⋅ACE2 complex in both, reduced (thiol groups) and

oxidized (disulfide bonds) states (Table 2) . Table 3 ).

However, when all disulfides in CoV/CoV-2 as well as ACE2 were reduced to thiols, the binding became thermodynamically unfavorable. In both cases, the binding free 

The study found that the reduction of all disulfides into sulfydryl groups completely impairs the binding of SARS-CoV/CoV-2 spike protein to ACE2. This is evident from the positive Gibbs energy of binding (∆ bind G o ) obtained for both CoV ox ⋅⋅⋅ACE2 ox and CoV-2 ox ⋅⋅⋅ACE2 ox complexes.

When the disulfides of only ACE2 were reduced to sulfydryl groups, the binding becomes weaker, as the The redox environment of cell surface receptors is regulated by the thiol-disulfide equilibrium in the extracellular region. 12, 18 This is maintained by glutathione transporters, 19 a number of oxidoreductases 12 including protein disulfide isomerase, 8 and several redox switches. 12 Under oxidative stress, the extracellular environment becomes oxidation-prone resulting more disulfide formation on protein surfaces. 12 Therefore, under severe oxidative stress, the cell surface receptor ACE2 and RBD of the intruding viral spike protein are likely to be present in its oxidized form having predominantly disulfide linkages. This computational study

shows that under oxidative stress, the lack of reducing environment would result in significantly favorable binding of the viral protein on the cell surface ACE2. In terms of energetics, this computational study demonstrates that the oxidized form of proteins with disulfide bridges would cause a 50 kcal/mol of decrease in Gibbs binding free energy. Furthermore, ACE2, which the viral spike proteins latch on to, is known to be a key player in the remedial of oxidative stress. 20 Binding of the viral protein will prevent the key catalytic function of ACE2 of converting angiotensin 2 (a strong activator of oxidative stress) to angiotensin 1-7 thereby creating a vicious circle of enhanced viral attack. In summary, the present study demonstrates 1 0 that the absence of or reduced oxidative stress would have a significant beneficial effect during early stage of viral infection by preventing viral protein binding on the host cells.

Computational Setup. Setting up of protein systems and all structural manipulations were carried out using Visual Molecular Dynamics (VMD). 21 Disulfide groups were modified to thiols during setting up of structures using standard VMD scripts. Molecular optimization and dynamics (MD) simulations were carried out using Nanoscale Molecular Dynamics (NAMD) package using CHARMM36 force field. [22] [23] [24] [25] [26] During MD simulations, electrostatic energy calculations were carried out using particle mesh Ewald method. 27 Backbone root-mean-squaredeviation (RMSD) calculations were performed using VMD. Protein-protein interactions were studied using Adaptive-Basis Poisson Boltzmann Solver (APBS). 28 Electrostatic field calculations were performed using PDB2PQR program suit. 29 Molecular Dynamics Simulations. All simulations were performed using the structure of ACE2 bound SARS-CoV (PDB entry: 3D0G) 1 3 and SCAR-CoV-2 (PDB entry: 6M0J) 15 . In all simulations, setup of protein complexes systems was carried out following protocols used previous studies from this lab. 17, 30 Briefly, hydrogens were added using the HBUILD module of CHARMM. Ionic amino acid residues were maintained in a protonation state corresponding to pH 7. The protonation state of histidine residues was determined by computing the pKa using Binding Free Energy Calculations. Gibbs free energies of binding between the ACE2 and SARS CoV or CoV-2 proteins were calculated using APBS using a standardized method of a treecode-accelerated boundary integral Poisson-Boltzmann equation solver (TABI-PB). 33 In this method, the protein surface is triangulated, and electrostatic surface potentials are computed. The discretization of surface potentials is utilized to compute the net energy due to solvation as well as electrostatic interactions between the two protein subunits, as outlined in thermodynamic scheme, Scheme 1. Following Scheme 1, the free energy of binding of the two protein fragments in water, can be expressed as a sum of two components (eq. 1)

where the ∆ Coul G represents the Coulombic (electrostatic) interactions between the proteins occurring at the protein-protein interface (Scheme 1) and ∆ ∆ solv G is the difference of the solvation energies between the complex and the corresponding free proteins:

However, the solvation calculation used only part of the entire spike protein as well as the ACE2, therefore ∆ bind G TABI-PB was calibrated by correcting ∆ ∆ solv G using experimentally known binding free energy of ACE2···CoV-2:

where K d is the experimental dissociation constant, which is equal to 37 nM. 16 The corrected free energy of the solvation

Using the ∆ G corr and eq. 4, the corrected binding free energy, ∆ bind G o of all protein complexes is expressed by

As shown in eq. 4, the combination of last two terms in eq. 5 is equal to ∆ ∆ solv G corr. Therefore, eq. 5 can be simplified as: Figure 1 . Sequence alignment (generated by Clustal Omega 35 ) between the receptor-binding domain of SARS-CoV and SARS-CoV2 proteins. The "*" represents the identical residues, ":" represents similar residues, and gap represents dissimilar residues. The cysteine residues are highlighted in yellow.

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK 60 CoV ------------------PFGEVFNATKFPSVYAWERKKISNCVADYSVLYNSTFFSTFK 42 *********:* *****:**:**************: *****

CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNS 120 CoV CYGVSATKLNDLCFSNVYADSFVVKGDDVRQIAPGQTGVIADYNYKLPDDFMGCVLAWNT 102 ***** ********:********::**:********** ************ ***:***: The medium-scale fluctuations are shown in green.

Coronavirus envelope protein: current knowledge

Coronaviruses: an overview of their replication and pathogenesis

Structure analysis of the receptor binding of 2019-nCoV

High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa

Inhibition of human immunodeficiency virus infection by agents that interfere with thiol-disulfide interchange upon virus-receptor interaction

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry

Significant redox insensitivity of the functions of the SARS-CoV spike glycoprotein: comparison with HIV envelope

Cell surface protein disulfide isomerase regulates natriuretic peptide generation of cyclic guanosine monophosphate

The role of cellular oxidoreductases in viral entry and virus infectionassociated oxidative stress: potential therapeutic applications

Implications of oxidative stress on viral pathogenesis

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

Thiol-Disulfide Exchange Reactions in the Mammalian Extracellular Environment

Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2

Crowder-Induced Conformational Ensemble Shift in Escherichia coli Prolyl-tRNA Synthetase

From structure to redox: The diverse functional roles of disulfides and implications in disease

Plasma membrane glutathione transporters and their roles in cell physiology and pathophysiology

ACE2-mediated reduction of oxidative stress in the central nervous system is associated with improvement of autonomic function

VMD: visual molecular dynamics

CHARMM Additive All-Atom Force Field for Glycosidic Linkages between Hexopyranoses

CHARMM additive all-atom force field for glycosidic linkages in carbohydrates involving furanoses

CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling

Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone (2) dihedral angles

CHARMM Additive All-Atom Force Field for Phosphate and Sulfate Linked to Carbohydrates

A smooth particle mesh Ewald method

Improvements to the APBS biomolecular solvation software suite

PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations

Multiple pathways promote dynamical coupling between catalytic domains in Escherichia coli prolyl-tRNA synthetase

Computer ""experiments" on classic fluids. I. Thermodynamical properties of Lennard-Jones molecules

Scalable molecular dynamics with NAMD

A treecode-accelerated boundary integral Poisson-Boltzmann solver for electrostatics of solvated biomolecules

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

The EMBL-EBI search and sequence analysis tools APIs in 2019

We acknowledge computational support from the Blugold Super Computing Cluster (BGSC) of University of Wisconsin-Eau Claire.

 Figure. 2. Structures of protein complexes of a) SARS-CoV⋅⋅⋅ACE2 and b) SARS-CoV-2⋅⋅⋅ACE2. All the disulfide bridges between cysteine residues are shown in green vdW spheres and thiol groups in cyan licorice.