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

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Abstract

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.

About the authors

K. M. Boyko

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, 119071, Moscow, Russia

Email: kmb@inbi.ras.ru
Россия, Москва

L. A. Varfolomeeva

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, 119071, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

N. A. Egorkin

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, 119071, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

M. E. Minyaev

Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia

Email: mminyaev@ioc.ac.ru
Россия, Москва

I. A. Alekseeva

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

I. A. Favorskaya

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

E. I. Ryabova

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

V. V. Prokofiev

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

I. B. Esmagambetov

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

D. V. Shcheblyakov

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

D. Yu. Logunov

Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

A. L. Gintsburg

FSBI National Research Centre for Epidemiology and Microbiology named after the honorary academician N.F. Gamaleya of the Ministry of Health of Russia

Email: gintsburg@gamaleya.org
ORCID iD: 0000-0003-1769-5059

Gintsburg, Alexander Leonidovich - Ph.D., D.Sci. (Biol.), Academician of RAS, Director-in-chief.

123098, Moscow

Russian Federation, Россия, Москва

V. O. Popov

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, 119071, Moscow, Russia

Email: nikolai.sluchanko@mail.ru
Россия, Москва

N. N. Sluchanko

Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology,” Russian Academy of Sciences, 119071, Moscow, Russia; Gamaleya National Research Center for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098, Moscow, Russia

Author for correspondence.
Email: nikolai.sluchanko@mail.ru
Россия, Москва; Россия, Москва

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