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

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.