Functional GPCR Expression in Eukaryotic LEXSY System

Aleksandra Luginina; Ivan Maslov; Polina Khorn; Oleksandr Volkov; Andrey Khnykin; Pavel Kuzmichev; Mikhail Shevtsov; Anatoliy Belousov; Ivan Kapranov; Dmitrii Dashevskii; Daniil Kornilov; Ekaterina Bestsennaia; Johan Hofkens; Jelle Hendrix; Thomas Gensch; Vadim Cherezov; Valentin Ivanovich; Alexey Mishin; Valentin Borshchevskiy

doi:10.1016/j.jmb.2023.168310

Functional GPCR Expression in Eukaryotic LEXSY System

J Mol Biol. 2023 Dec 1;435(23):168310. doi: 10.1016/j.jmb.2023.168310. Epub 2023 Oct 6.

Authors

Aleksandra Luginina¹, Ivan Maslov², Polina Khorn¹, Oleksandr Volkov³, Andrey Khnykin¹, Pavel Kuzmichev¹, Mikhail Shevtsov¹, Anatoliy Belousov¹, Ivan Kapranov¹, Dmitrii Dashevskii¹, Daniil Kornilov¹, Ekaterina Bestsennaia¹, Johan Hofkens⁴, Jelle Hendrix⁵, Thomas Gensch⁶, Vadim Cherezov⁷, Valentin Ivanovich¹, Alexey Mishin¹, Valentin Borshchevskiy⁸

Affiliations

¹ Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia.
² Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia; Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, Diepenbeek, Belgium; Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium.
³ No affiliation.
⁴ Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium; Max Planck Institute for Polymer Research, Mainz, Germany.
⁵ Dynamic Bioimaging Lab, Advanced Optical Microscopy Centre, Biomedical Research Institute, Agoralaan C (BIOMED), Hasselt University, Diepenbeek, Belgium; Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium.
⁶ Laboratory for Photochemistry and Spectroscopy, Division for Molecular Imaging and Photonics, Department of Chemistry, KU Leuven, Leuven, Belgium.
⁷ Bridge Institute, Department of Chemistry, University of Southern California, Los Angeles, CA, USA.
⁸ Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russia; Joint Institute for Nuclear Research, Dubna, Russia. Electronic address: borshchevskiy.vi@phystech.edu.

PMID: 37806553
DOI: 10.1016/j.jmb.2023.168310

Abstract

G protein-coupled receptors (GPCRs) form the largest superfamily of membrane proteins in the human genome, and represent one of the most important classes of drug targets. Their structural studies facilitate rational drug discovery. However, atomic structures of only about 20% of human GPCRs have been solved to date. Recombinant production of GPCRs for structural studies at a large scale is challenging due to their low expression levels and stability. Therefore, in this study, we explored the efficacy of the eukaryotic system LEXSY (Leishmania tarentolae) for GPCR production. We selected the human A_2A adenosine receptor (A_2AAR), as a model protein, expressed it in LEXSY, purified it, and compared with the same receptor produced in insect cells, which is the most popular expression system for structural studies of GPCRs. The A_2AAR purified from both expression systems showed similar purity, stability, ligand-induced conformational changes and structural dynamics, with a remarkably higher protein yield in the case of LEXSY expression. Overall, our results suggest that LEXSY is a promising platform for large-scale production of GPCRs for structural studies.

Keywords: G protein-coupled receptors; Leishmania tarentolae; Protein biophysical characterization; Recombinant expression; Single-molecule Förster Resonance Energy Transfer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Drug Discovery
Humans
Leishmania
Ligands
Protein Conformation
Protein Stability
Receptor, Adenosine A2A* / biosynthesis
Receptor, Adenosine A2A* / chemistry
Receptors, G-Protein-Coupled* / biosynthesis
Receptors, G-Protein-Coupled* / chemistry
Recombinant Proteins* / biosynthesis
Recombinant Proteins* / chemistry

Substances

Receptors, G-Protein-Coupled
Recombinant Proteins
Receptor, Adenosine A2A
Ligands