Circular dichroism studies of extracellular domains of human nicotinic acetylcholine receptors provide an insight into their structure.
The extracellular domains (ECDs) of human nicotinic acetylcholine receptors (nAChRs) are of major pharmacological interest as drug targets in the autoimmune disease myasthenia gravis and in various neurological disorders. We have previously expressed and purified the human muscle alpha1-, beta1-, gamma- and epsilon-nAChR-ECDs, as well as the wild type and a mutant of neuronal alpha7-ECD, in yeast Pichia pastoris. The far-UV circular dichroism (CD) studies of these ECDs, presented here, revealed a major prevalence of beta-sheet ( approximately 40%) and a small proportion of alpha-helical ( approximately 5%) structure for all ECDs, in good agreement with the secondary structure composition of the Torpedo muscle-type nAChR-ECDs and in less, but considerable, agreement with that of the homologous invertebrate acetylcholine-binding proteins (AChBPs). The near-UV CD studies of these nAChR-ECDs indicated well-defined tertiary structures, as was previously suggested by biochemical and immunochemical studies. Furthermore, the binding of cholinergic ligands to the mutant of alpha7-ECD resulted in no changes in its secondary structure, but revealed significant local conformational changes. Our present studies probe the structure of human nAChR-ECDs for the first time and further suggest that our expressed proteins fold to a near-native conformation, thus being suitable for further structural studies.
Department of Biochemistry, Hellenic Pasteur Institute, 127, Vas. Sofias Ave., GR11521 Athens, Greece.
Marios
M
Zouridakis
Kalliopi
K
Kostelidou
Alexandros
A
Sotiriadis
Christos
C
Stergiou
Elias
E
Eliopoulos
Konstantinos
K
Poulas
Socrates J
SJ
Tzartos
D000595:Amino Acid Sequence; D002038:Bungarotoxins / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002942:Circular Dichroism / Q000379:methods*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005822:Genetic Vectors; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D002038:Bungarotoxins; D018076:DNA, Complementary; D008024:Ligands; D011978:Receptors, Nicotinic; D002217:Carbachol