Preparation and Crystallographic Analysis of a Complex of SARS-CoV-2 S-Protein Receptor-Binding Domain with a Virus-Neutralizing Nanoantibody

• Published in: Crystallography reports Vol. 68; no. 6; pp. 864 - 871
• Main Authors: Boyko, K. M., Varfolomeeva, L. A., Egorkin, N. A., Minyaev, M. E., Alekseeva, I. A., Favorskaya, I. A., Ryabova, E. I., Prokofiev, V. V., Esmagambetov, I. B., Shcheblyakov, D. B., Logunov, D. Yu, Gintsburg, A. L., Popov, V. O., Sluchanko, N. N.
• Format: Journal Article
• Language: English
• Published: Moscow Pleiades Publishing 01-12-2023; Springer Nature B.V
• Subjects: Antibodies; Binding; COVID-19; Crystal growth; Crystallography; Crystallography and Scattering Methods; Gel filtration; Glycoproteins; Heterogeneity; Neutralizing; Physics; Physics and Astronomy; Proteins; Radiation sources; Receptors; Severe acute respiratory syndrome coronavirus 2; Structure of Macromolecular Compounds; Synchrotron radiation; Unit cell; Viral diseases; Viruses
• ISSN: 1063-7745; 1562-689X
• DOI: 10.1134/S1063774523601168

Coronavirus SARS-CoV-2 was the cause of global pandemic COVID-19 that has claimed millions of lives around the world. One of the approaches to the therapy of severe forms of COVID-19 is based on the use of virus-neutralizing antibodies against the receptor-binding domain (RBD) of the virus spike glycoprotein (S). Detailed structural data on particular epitopes recognized by these virus-neutralizing antibodies is an important aspect of the improvement of available antibodies in order to extend the range of recognized virus strains. A stoichiometric complex of the virus-neutralizing single-domain antibody P2C5 and the glycosylated form of SARS-CoV-2 S protein RBD was obtained. Despite numerous conditions in which the crystal growth of the complex was observed, these crystals showed poor diffraction, apparently because of heterogeneity associated with the glycosylation of RBD. With the aim of improving the crystal quality, deglycosylase PNGase F was produced in a bacterial expression system and deglycosylation of RBD was performed. Despite low solubility of the deglycosylated form of RBD, it retains the ability to bind to the nanoantibody P2C5, resulting in the formation of a stable heterodimeric complex during gel filtration. The crystals of this complex were obtained. They showed diffraction to a similar resolution of ~4 Å on a laboratory X-ray diffractometer and at a synchrotron radiation source. The symmetry space group, the unit cell parameters of the crystal, and its protein composition were determined.