Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties

Biochim Biophys Acta. 2009 Feb;1794(2):355-66. doi: 10.1016/j.bbapap.2008.11.002. Epub 2008 Nov 19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bungarotoxins / chemistry
  • Glycosylation
  • Humans
  • Ligands
  • Mice
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Nicotine / chemistry
  • Nicotinic Agonists / chemistry
  • Nicotinic Antagonists / chemistry
  • Pichia / metabolism
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Radioligand Assay
  • Receptors, Nicotinic / biosynthesis*
  • Receptors, Nicotinic / chemistry*
  • Receptors, Nicotinic / genetics
  • Solubility
  • Torpedo
  • Tubocurarine / chemistry
  • alpha7 Nicotinic Acetylcholine Receptor

Substances

  • Bungarotoxins
  • Chrna7 protein, human
  • Chrna7 protein, mouse
  • Ligands
  • Nicotinic Agonists
  • Nicotinic Antagonists
  • Receptors, Nicotinic
  • alpha7 Nicotinic Acetylcholine Receptor
  • Nicotine
  • Tubocurarine