International Journal of Biomedicine.2019;9 Suppl_1:S19-S19.
Originally published June 29, 2019
Background: Nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel, which is widely distributed both in the central and peripheral nervous system, and in some of the non-neuronal tissues, including epithelium and immune cells. The pathophysiology of a number of diseases is associated with dysfunctions of this receptor, including neurodegenerative and mental disorders like Alzheimer disease (AD) and schizophrenia. The nicotinic receptor of α7 type (α7-nAChR) plays important role in the memory and learning processes and is inhibited by soluble aggregates of β-amyloid peptide (Aβ). Aβ1-42 is the most toxic form of the amyloid peptide.
Methods: The water-soluble analogue of the ligand-binding extracellular domain of α7-nAChR was produced in Pichia pastoris and purified from a culture medium by Ni-NTA and size-exclusion chromatography. The equilibrium dissociation constant (Kd) of the complex of the recombinant domain with α-bungarotoxin was measured on the optical SPR biosensor Biacore 3000. Structures of the α7 domain alone and in the complex with oligomeric Aβ1-42 peptide were studied by cryoelectron microscopy (cryo-EM).
Results: The α7 ligand-binding domain has an increased stability in solution, and demonstrates ligand-binding characteristics similar to those of the native receptor (Kd of the domain/α-bungarotoxin complex 28±2nM). Statistical analysis of the cryo-EM images of the individual domain particles revealed the presence of a pentameric structure, confirming intact subunit assembly. Unfortunately, the domain demonstrated the preferable orientation on grids with the top view. Nevertheless, the 3D structure of the domain with a height ~ 7 nm, external diameter of ~ 9 nm, and the pore diameter of ~ 2 nm was reconstructed at 8.5 Å resolution. 2D classification of the cryo-EM images of the domain particles in the complex with Aβ1-42 revealed the conformational changes appeared due to interaction with the amyloid peptide.
Conclusion: Obtained results open new perspectives for structural studies of the nAChR ligand-binding domains in complex with the ligands which escape crystallization.