Citation

  • Authors: Grishin AM. et al.
  • Year: 2022
  • Journal: J Mol Biol 434 167357
  • Applications: in vitro / DNA / FectoPRO
  • Cell type: Expi293F
    Description: Human embryonic kidney Fibroblast
    Known as: Expi 293-F, Expi, HEK-293 Expi

Method

Cells: Expi293F Growth medium: Gibco Expi293 Expression Medium (at 37 °C at 125 rpm, humidified atmosphere and 8% CO2) Seeding: density of 3 × 106 cells/ml Transfection conditions: 1 µg of plasmid DNA per 1 ml of cell culture and FectoPro® Transfection enhancers were added the next day. The media was harvested 4 days after transfection, cleared by centrifugation at 20,000xg for 1 hour at +4 °C, filtered through a 0.22 µm filter and applied for further purification.

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.

Pubmed