期刊
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
卷 1794, 期 2, 页码 355-366出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbapap.2008.11.002
关键词
Human alpha 7 nicotinic acetylcholine receptor extracellular domain; 3D model; Circular dichroism spectroscopy; Ligand-binding; Dynamic light scattering; Electron microscopy
资金
- EU-European
- Greek Ministry of Development-GSRT
- EC FP7 [202088]
In order to facilitate structural studies of the extracellular domain (ECD) of human alpha 7 nicotinic acetylcholine receptor (nAChR), we designed several mutants, since the wild-type-ECD forms large oligomers and microaggregates, and expressed them in the yeast Pichia pastoris. Mutant design was based on a 3D model of human alpha 7-nAChR-ECD, constructed using as templates the X-ray crystal structure of the homologous acetylcholine-binding protein (AChBP) and the electron microscopy structure of the Torpedo alpha-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Va169Thr, Cys116Ser, Ile165Thr, Val177Thr, Phe187Tyr) and the replacement of its Cys-loop with the corresponding and more hydrophilic AChBP Cys-loop, was expressed with a 4-fold higher expression yield (1.2 mg/L) than the wild-type alpha 7-ECD, existing exclusively as it soluble oligomeric, probably pentameric, form, at concentrations up to at least 10 mg/mL, as judged by gel filtration and dynamic light scattering. This mutant displayed a significantly improved I-125-alpha-bungarotoxin-binding affinity (K-d=24 nM) compared to the wild-type-ECD (K-d=70 nM), the binding being inhibited by unlabelled alpha-bungarotoxin, D-tubocurarine or nicotine (K-i of 21.5 nM, 127 mu M and 17.5 mM, respectively). Circular dichroism, studies of mut10 revealed (a) a similar secondary structure composition (similar to 5% alpha-helix, similar to 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha 1-nAChR-ECD, (b) a well-defined tertiary structure and (c) binding of small cholinergic ligands at micromolar concentrations. Furthermore, electron microscopy showed well-assembled, probably pentameric. particles of mut10. Finally, since deglycosylation did not alter its solubility or ligand-binding properties, mut10, in either its glycosylated or deglycosylated form, is a promising alpha 7-ECD mutant for structural studies, useful for the rational drug design to treat alpha 7-nAChR-related diseases. (C) 2008 Elsevier B.V. All rights reserved.
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