Linked Life Data

19059502

Source:http://linkedlifedata.com/resource/pubmed/id/19059502

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PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
2009-1-12
pubmed:abstractText
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.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-3002
pubmed:author
pubmed:issnType
Print
pubmed:volume
1794
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
355-66
pubmed:dateRevised
2010-6-4
pubmed:meshHeading
pubmed-meshheading:19059502-Amino Acid Sequence, pubmed-meshheading:19059502-Animals, pubmed-meshheading:19059502-Bungarotoxins, pubmed-meshheading:19059502-Glycosylation, pubmed-meshheading:19059502-Humans, pubmed-meshheading:19059502-Ligands, pubmed-meshheading:19059502-Mice, pubmed-meshheading:19059502-Models, Molecular, pubmed-meshheading:19059502-Molecular Sequence Data, pubmed-meshheading:19059502-Mutation, pubmed-meshheading:19059502-Nicotine, pubmed-meshheading:19059502-Nicotinic Agonists, pubmed-meshheading:19059502-Nicotinic Antagonists, pubmed-meshheading:19059502-Pichia, pubmed-meshheading:19059502-Protein Structure, Secondary, pubmed-meshheading:19059502-Protein Structure, Tertiary, pubmed-meshheading:19059502-Radioligand Assay, pubmed-meshheading:19059502-Receptors, Nicotinic, pubmed-meshheading:19059502-Solubility, pubmed-meshheading:19059502-Torpedo, pubmed-meshheading:19059502-Tubocurarine
pubmed:year
2009
pubmed:articleTitle
Design and expression of human alpha7 nicotinic acetylcholine receptor extracellular domain mutants with enhanced solubility and ligand-binding properties.
pubmed:affiliation
Department of Biochemistry, Hellenic Pasteur Institute, 127, GR11521, Athens, Greece.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't