title: Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.
abstract: In order to facilitate structural studies of the extracellular domain (ECD) of human alpha7 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 alpha7-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, 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 alpha7-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 (125)I-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 microM and 17.5 mM, respectively). Circular dichroism studies of mut10 revealed (a) a similar secondary structure composition ( approximately 5% alpha-helix, approximately 45% beta-sheet) to that of the AChBP, Torpedo alpha-nAChR-ECD, and mouse alpha1-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 alpha7-ECD mutant for structural studies, useful for the rational drug design to treat alpha7-nAChR-related diseases.
authors.affiliation: Department of Biochemistry, Hellenic Pasteur Institute, 127, GR11521, Athens, Greece.
authors.forename: Marios
authors.initials: M
authors.lastname: Zouridakis
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Zisimopoulou
authors.forename: Elias
authors.initials: E
authors.lastname: Eliopoulos
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Poulas
authors.forename: Socrates J
authors.initials: SJ
authors.lastname: Tzartos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002038:Bungarotoxins / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009538:Nicotine / Q000737:chemistry; D018722:Nicotinic Agonists / Q000737:chemistry; D018733:Nicotinic Antagonists / Q000737:chemistry; D010843:Pichia / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D014101:Torpedo; D014403:Tubocurarine / Q000737:chemistry; D064569:alpha7 Nicotinic Acetylcholine Receptor
chemical_list: D002038:Bungarotoxins; C579167:Chrna7 protein, human; C579166:Chrna7 protein, mouse; D008024:Ligands; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine; D014403:Tubocurarine