Efficient screening of ligand-receptor complex formation using fluorescence labeling and size-exclusion chromatography

• Published in: Biochemical and biophysical research communications 2020-10, Vol.532 (1), p.127-133
• Main Authors: Kulbatskii, D.S., Shulepko, M.A., Sluchanko, N.N., Yablokov, E.O., Kamyshinsky, R.A., Chesnokov, Y.M., Kirpichnikov, M.P., Lyukmanova, E.N.
• Format: Article
• Language: English
• Subjects: alpha7 Nicotinic Acetylcholine Receptor - chemistry; alpha7 Nicotinic Acetylcholine Receptor - metabolism; alpha7 Nicotinic Acetylcholine Receptor - ultrastructure; Animals; Bungarotoxins - chemistry; Bungarotoxins - metabolism; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Carrier Proteins - ultrastructure; Chromatography, Gel; Cryo-EM; Cryoelectron Microscopy; Elapid Venoms - chemistry; Elapid Venoms - metabolism; Fluorescent Dyes; Humans; Ligands; Ly6/uPAR; Nicotinic acetylcholine receptor; Protein Interaction Domains and Motifs; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - ultrastructure; Surface Plasmon Resonance; Three-finger protein; α-neurotoxins
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2020.08.021

Evidence of a complex formation is a crucial step in the structural studies of ligand-receptor interactions. Here we presented a simple and fast approach for qualitative screening of the complex formation between the chimeric extracellular domain of the nicotinic acetylcholine receptor (α7-ECD) and three-finger proteins. Complex formation of snake toxins α-Bgtx and WTX, as well as of recombinant analogs of human proteins Lynx1 and SLURP-1, with α7-ECD was confirmed using fluorescently labeled ligands and size-exclusion chromatography with simultaneous absorbance and fluorescence detection. WTX/α7-ECD complex formation also was confirmed by cryo-EM. The proposed approach could easily be adopted to study the interaction of other receptors with their ligands. •SPR is not always applicable for analyzing the ligand-receptor interaction.•SEC with fluorescent ligands can be used for study of ligand-receptor interaction.•Complexes of three-finger proteins with α7 nicotinic receptor domain were analyzed.•Complex formation was proven by cryoelectronic microscopy.