Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor
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...
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International Medical Research and Development Corporation
2019-06-01
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doaj-84f7a7ca9e624f188bd44822b71bea442020-11-25T00:40:30ZengInternational Medical Research and Development CorporationInternational Journal of Biomedicine2158-05102158-05292019-06-019Suppl_1S19S1910.21103/IJBM.9.Suppl_1.P7Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine ReceptorAndrey V. Tsarev0Roman A. Kamyshinsky1Vasiliy I. Mikirtumov2Dmitriy S. Kulbatskii3Eugene O. Yablokov4Zakhar O. Shenkarev5Olga S. Sokolova6Ekaterina N. Lyukmanova7Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russian FederationNational Research Center, Kurchatov Institute, Moscow, RussiaLomonosov Moscow State University, Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, RussiaInstitute of Biomedical Chemistry, Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russian FederationLomonosov Moscow State University, Moscow, RussiaShemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia; Moscow Institute of Physics and Technology, Dolgoprudny, Moscow Region, Russian FederationBackground: 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.http://ijbm.org/articles/IJBM_2019_9_S1_P7.pdfcryo-electron microscopyα7-nAChRbeta-amyloid peptide |
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DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Andrey V. Tsarev Roman A. Kamyshinsky Vasiliy I. Mikirtumov Dmitriy S. Kulbatskii Eugene O. Yablokov Zakhar O. Shenkarev Olga S. Sokolova Ekaterina N. Lyukmanova |
spellingShingle |
Andrey V. Tsarev Roman A. Kamyshinsky Vasiliy I. Mikirtumov Dmitriy S. Kulbatskii Eugene O. Yablokov Zakhar O. Shenkarev Olga S. Sokolova Ekaterina N. Lyukmanova Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor International Journal of Biomedicine cryo-electron microscopy α7-nAChR beta-amyloid peptide |
author_facet |
Andrey V. Tsarev Roman A. Kamyshinsky Vasiliy I. Mikirtumov Dmitriy S. Kulbatskii Eugene O. Yablokov Zakhar O. Shenkarev Olga S. Sokolova Ekaterina N. Lyukmanova |
author_sort |
Andrey V. Tsarev |
title |
Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor |
title_short |
Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor |
title_full |
Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor |
title_fullStr |
Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor |
title_full_unstemmed |
Abstract P-7: Cryoelectron Microscopy Study of Water-Soluble Extracellular Domain of α7 Nicotinic Acetylcholine Receptor |
title_sort |
abstract p-7: cryoelectron microscopy study of water-soluble extracellular domain of α7 nicotinic acetylcholine receptor |
publisher |
International Medical Research and Development Corporation |
series |
International Journal of Biomedicine |
issn |
2158-0510 2158-0529 |
publishDate |
2019-06-01 |
description |
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. |
topic |
cryo-electron microscopy α7-nAChR beta-amyloid peptide |
url |
http://ijbm.org/articles/IJBM_2019_9_S1_P7.pdf |
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