Design and expression of human α7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties

dc.contributor.author	Zouridakis, M	en
dc.contributor.author	Zisimopoulou, P	en
dc.contributor.author	Eliopoulos, E	en
dc.contributor.author	Poulas, K	en
dc.contributor.author	Tzartos, SJ	en
dc.date.accessioned	2014-06-06T06:49:39Z
dc.date.available	2014-06-06T06:49:39Z
dc.date.issued	2009	en
dc.identifier.issn	15709639	en
dc.identifier.uri	http://dx.doi.org/10.1016/j.bbapap.2008.11.002	en
dc.identifier.uri	http://62.217.125.90/xmlui/handle/123456789/4702
dc.subject	3D model	en
dc.subject	Circular dichroism spectroscopy	en
dc.subject	Dynamic light scattering	en
dc.subject	Electron microscopy	en
dc.subject	Human α7 nicotinic acetylcholine receptor extracellular domain	en
dc.subject	Ligand-binding	en
dc.subject.other	acetylcholine binding protein	en
dc.subject.other	alpha bungarotoxin	en
dc.subject.other	binding protein	en
dc.subject.other	nicotine	en
dc.subject.other	nicotinic acetylcholine receptor alpha 7	en
dc.subject.other	nicotinic receptor	en
dc.subject.other	tubocurarine chloride	en
dc.subject.other	unclassified drug	en
dc.subject.other	article	en
dc.subject.other	binding affinity	en
dc.subject.other	circular dichroism	en
dc.subject.other	controlled study	en
dc.subject.other	crystal structure	en
dc.subject.other	deglycosylation	en
dc.subject.other	electron microscopy	en
dc.subject.other	gel filtration	en
dc.subject.other	glycosylation	en
dc.subject.other	human	en
dc.subject.other	hydrophilicity	en
dc.subject.other	ligand binding	en
dc.subject.other	light scattering	en
dc.subject.other	Pichia pastoris	en
dc.subject.other	point mutation	en
dc.subject.other	priority journal	en
dc.subject.other	protein expression	en
dc.subject.other	protein structure	en
dc.subject.other	sequence alignment	en
dc.subject.other	Amino Acid Sequence	en
dc.subject.other	Animals	en
dc.subject.other	Bungarotoxins	en
dc.subject.other	Glycosylation	en
dc.subject.other	Humans	en
dc.subject.other	Ligands	en
dc.subject.other	Mice	en
dc.subject.other	Models, Molecular	en
dc.subject.other	Molecular Sequence Data	en
dc.subject.other	Mutation	en
dc.subject.other	Nicotine	en
dc.subject.other	Nicotinic Agonists	en
dc.subject.other	Nicotinic Antagonists	en
dc.subject.other	Pichia	en
dc.subject.other	Protein Structure, Secondary	en
dc.subject.other	Protein Structure, Tertiary	en
dc.subject.other	Radioligand Assay	en
dc.subject.other	Receptors, Nicotinic	en
dc.subject.other	Solubility	en
dc.subject.other	Torpedo	en
dc.subject.other	Tubocurarine	en
dc.subject.other	Pichia pastoris	en
dc.title	Design and expression of human α7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties	en
heal.type	journalArticle	en
heal.identifier.primary	10.1016/j.bbapap.2008.11.002	en
heal.publicationDate	2009	en
heal.abstract	In order to facilitate structural studies of the extracellular domain (ECD) of human α7 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 α7-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 α-nAChR-ECD. At least one mutant, mut10, carrying six single-point mutations (Phe3Tyr, Val69Thr, 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 α7-ECD, existing exclusively as a 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 125I-α-bungarotoxin-binding affinity (Kd = 24 nM) compared to the wild-type-ECD (Kd = 70 nM), the binding being inhibited by unlabelled α-bungarotoxin, d-tubocurarine or nicotine (Ki of 21.5 nM, 127 μM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition (∼ 5% α-helix, ∼ 45% β-sheet) to that of the AChBP, Torpedo α-nAChR-ECD, and mouse α1-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 α7-ECD mutant for structural studies, useful for the rational drug design to treat α7-nAChR-related diseases. © 2008 Elsevier B.V. All rights reserved.	en
heal.journalName	Biochimica et Biophysica Acta - Proteins and Proteomics	en
dc.identifier.issue	2	en
dc.identifier.volume	1794	en
dc.identifier.doi	10.1016/j.bbapap.2008.11.002	en
dc.identifier.spage	355	en
dc.identifier.epage	366	en