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Originally published In Press as doi:10.1074/jbc.M108845200 on January 25, 2002

J. Biol. Chem., Vol. 277, Issue 15, 12613-12621, April 12, 2002
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Yeast Expression and NMR Analysis of the Extracellular Domain of Muscle Nicotinic Acetylcholine Receptor alpha  Subunit*

Yun YaoDagger , Junmei WangDagger , Nitnara ViroonchatapanDagger , Avraham Samson§, Jordan Chill§, Elizabeth RotheDagger , Jacob Anglister§, and Zuo-Zhong WangDagger ||

From the Dagger  Department of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261 and the § Department of Structural Biology, the Weizmann Institute of Science, Rehovot 76100, Israel

The alpha  subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for alpha -bungarotoxin and for autoimmune antibodies in sera of patients with myasthenia gravis. To obtain sufficient materials for structural studies of the receptor-ligand complexes, we have expressed part of the mouse muscle alpha  subunit as a soluble, secretory protein using the yeast Pichia pastoris. By testing a series of truncated fragments of the receptor protein, we show that alpha 211, the entire amino-terminal extracellular domain of AChR alpha  subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The alpha 211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with N-linked glycosylation on SDS-polyacrylamide gel. The protein was purified to homogeneity by isoelectric focusing electrophoresis (pI 5.8), and it appeared as a 4.5 S monomer on sucrose gradient at concentrations up to 1 mM (~30 mg/ml). The receptor domain bound monoclonal antibody mAb35, a conformation-specific antibody against the main immunogenic region of the AChR. In addition, it formed a high affinity complex with alpha -bungarotoxin (kD 0.2 nM) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of alpha 211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently 15N-labeled in P. pastoris, and proton cross-peaks were well dispersed in nuclear Overhauser effect and heteronuclear single quantum coherence spectra as measured by NMR spectroscopy. We conclude that the soluble AChR protein is useful for high resolution structural studies.

* This work was supported by National Institutes of Health Grant NS38301, the Muscular Dystrophy Association, a Competitive Medical Research Fund award from the University of Pittsburgh (to Z.-Z. W.), and by United States-Israel Binational Science Foundation Grant 98-328 (to J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Joseph and Ruth Owades Professor in chemistry.

|| To whom correspondence should be addressed: Dept. of Neurobiology, University of Pittsburgh School of Medicine, 3500 Terrace St., E1440 BST, Pittsburgh, PA 15261. Tel.: 412-648-9421; Fax: 412-383-8663; E-mail: zzwang@pitt.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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