Research Article
Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen

https://doi.org/10.1016/j.jmb.2021.167357Get rights and content

Highlights

  • The Receptor-Binding Domain of the Spike protein is dependent on its disulfide bonds.

  • Reduction of the disulfide bonds in the RBD render it unstable.

  • Reduction of the disulfide bonds of the RBD decreases its affinity to ACE2.

  • The highly-reducing environment can stop SARS-CoV-2 replication in cell-based assays.

Abstract

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456–490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36–39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function.

Abbreviations

ACE2
Angiotensin-converting Enzyme 2
CD
Circular Dichroism
cryo-EM
Cryo-Electron Microscopy
CV
Column Volume
DMEM
Dulbecco's Modified Eagle Medium
DPP4
Dipeptidyl Peptidase-4
DTT
Dithiothreitol
EC90
Effective Concentration 90%
ELF
Epithelial Lining Fluid
FBS
Fetal Bovine Serum
GSH
Reduced Glutathione
hAPN
Human Aminopeptidase N
HCoV
Human Coronavirus
MERS
Middle East Respiratory Syndrome
MST
Microscale Thermophoresis
MTS
5-(3-carboxymethoxyphenyl)-2-(4,5-dimethyl-thiazoly)-3-(4-sulfophenyl) tetrazolium
NAC
N-Acetyl-L-cysteine
PCR
Polymerase Chain Reaction
RBD
Receptor-binding Domain
RMSD
Root Mean Square Deviation
RMSF
Root Mean Square Fluctuation
RT
Room Temperature
SARS
Severe Acute Respiratory Syndrome
TCEP
tris(2-carboxyethyl)phosphine
TCID50
Tissue Culture 50% Infectious Dose

Keywords

SARS-CoV-2
disulfide-reducing agent
disulfide bond
receptor-binding domain
RBD

Data availability

We have shared the link to molecular dynamics data in the text of the main manuscript and revision notes. The DOI link will become active upon acceptance of the manuscript.

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