Yeast Expression and NMR Analysis of the Extracellular Domain of Muscle Nicotinic Acetylcholine Receptor α Subunit

Abstract

The ␣ subunit of the nicotinic acetylcholine receptor (AChR) from Torpedo electric organ and mammalian muscle contains high affinity binding sites for ␣-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 ␣ 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 ␣211, the entire amino-terminal extracellular domain of AChR ␣ subunit (amino acids 1-211), is the minimal segment that could fold properly in yeast. The ␣211 protein was secreted into the culture medium at a concentration of >3 mg/liter. It migrated as a 31-kDa polypeptide with Nlinked 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 (ϳ30 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 ␣-bungarotoxin (k D 0.2 nM) but showed relatively low affinity to the small cholinergic ligand acetylcholine. Circular dichroism spectroscopy of ␣211 revealed a composition of secondary structure corresponding to a folded protein. Furthermore, the receptor fragment was efficiently 15 N-labeled in P. pastoris, and proton crosspeaks 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.The nicotinic AChRs 1 are members of the superfamily of ligand-gated ion channels that mediate fast ion flow across postsynaptic membranes in neural and muscle cells. This family of proteins includes the receptors for glycine, ␥-aminobutyric acid, glutamate, and serotonin (1, 2). The AChR from Torpedo electric organ and skeletal muscle is a large (ϳ290 kDa) heteropentameric complex consisting of four homologous subunits in the stoichiometry of ␣ 2 ␤␦␥ (Torpedo and embryonic muscle) or ␣ 2 ␤␦⑀ (adult muscle (3, 4)). The subunits are arranged in the order ␣-␥-␣-␦-␤ to create a cylindrical complex around the ion channel (5-7). Each subunit is a single polypeptide chain that has a large extracellular amino-terminal domain followed by three transmembrane domains, a long intracellular loop, a fourth transmembrane domain, and a short extracellular carboxyl-terminal tail (8 -10). The large extracellular domain of the AChR confers high affinity binding activity for major agonists and competitive antagonists. The neurotransmitter ACh interacts with two nonequivalent sites near the interface of the ␣␦ and ␣␥ subunits (2, 11, 12). The binding site for ␣-bungarotoxin (␣-BuTx) is located largely on the amino-terminal extracellular domain of the ␣ subunit (13-16). In additio...

Solution Conformation of αA-conotoxin EIVA, a Potent Neuromuscular Nicotinic Acetylcholine Receptor Antagonist from Conus ermineus

“…The second feature that emerged from structural studies of ␣/␣A-conotoxins is the estimated size of the ligand binding pocket in nAChR (15). The suggested size of the ligand binding pocket of ϳ20 Å (height) ϫ 20 Å (width) ϫ 15 Å (thickness) is in good agreement with what has been proposed by electromagnetic images (38,39) or by a recent homology-modeling study (51). ␣A-Conotoxins are larger than typical ␣-conotoxins and hence may need to bend or deform themselves to fit into the same ligand binding pocket of nAChR.…”

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

“…CD and FTIR spectra further indicated significant amounts of ␤-sheet in the extracellular domain (48% by FTIR investigation), in good agreement with values found for the acetylcholine binding protein (14) and AChR extracellular domains (Table IV). However, low ␣-helical contents of 8% were found for the AChBP from Lymnaea stagnalis (14), and 12-14% for soluble, monomeric preparations of mouse muscle nAChR N-terminal domains (38,39). In contrast, preparations of rat ␣7 and Torpedo N-terminal AChR domains, both forming oligomeric assemblies, showed ␣-helical contents of 32-44% (13, 36).…”

Crystal Structure of the Extracellular Domain of a Bacterial Ligand-Gated Ion Channel

“…The best result was achieved with the α1-nAChR subunit, which does not form homopentamers in vivo but assembles into α1-γ-α1-δ-β heteropentamers. The initial expression of the α1-ECD in a monomeric state 16 was improved by the selection of a triple mutant with increased solubility, whose structure could be solved in complex with α-bungarotoxin. 17 The discovery of bacterial members in the pLGIC family, 18 followed by the functional characterization of one of them, 19 recently changed this picture dramatically, leading to the crystallization and resolution of the X-ray structures of entire proteins of this family.…”