The Emerging Three‐Dimensional Structure of a Receptor

Hucho, Ferdinand; Tsetlin, Victor I.; Machold, Jan

doi:10.1111/j.1432-1033.1996.0539u.x

Cited by 203 publications

(210 citation statements)

References 187 publications

(170 reference statements)

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“…It is an allosteric and integral membrane protein composed of four different subunits arranged pseudo-pentamerically in the stoichiometry of 2␣1:␤1:␦ or ⑀:␥ to form an ion channel. Each subunit contains a large hydrophilic amino-terminal (NH 2 ) domain that faces the extracellular environment, four transmembrane domains (M1, M2, M3, and M4) made up of 19 -25 amino acids, a large cytoplasmic loop between the M3 and M4 domains, and a short extracellular carboxylic terminal (COOH) domain (1)(2)(3).…”

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confidence: 99%

Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Oyola-Cintrón

Caballero-Rivera

Ballester

et al. 2015

Journal of Biological Chemistry

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confidence: 99%

Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Oyola-Cintrón

Caballero-Rivera

Ballester

et al. 2015

Journal of Biological Chemistry

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“…Our results confirm that the N-terminal extracellular domain indeed harbors major elements of the ligand recognition function of the nAChR, as has long been suggested on the basis of affinity labeling and immunological studies (for recent reviews, see Refs. 1,2,8,9,15,and 16). …”

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confidence: 99%

Expression and Renaturation of the N-terminal Extracellular Domain of Torpedo Nicotinic Acetylcholine Receptor α-Subunit

Schrattenholz¹,

Pfeiffer²,

Pejovic³

et al. 1998

Journal of Biological Chemistry

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The N-terminal extracellular region (amino acids 1-209) of the ␣-subunit of the nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata electric tissue was expressed as inclusion bodies in Escherichia coli using the pET 3a vector. Employing a novel protocol of unfolding and refolding, in the absence of detergent, a water-soluble globular protein of 25 kDa was obtained displaying approximately 15% ␣-helical and 45% ␤-structure. The fragment bound ␣- So far, most of our knowledge on the three-dimensional structure of neuroreceptors is based on a combination of electronmicroscopical, biochemical and immunological data obtained for the nicotinic acetylcholine receptor (nAChR) 1 from the electric ray Torpedo (1, 2). These studies have elucidated the overall dimensions of the receptor protein (3), its position with respect to the surrounding lipid bilayer, the locations of functional domains and amino acid residues belonging to the integral ion channel (4, 5), its gating structures (6, 7), and the binding sites for several classes of ligands (1, 2, 7-12). However, a high resolution three-dimensional structure of the nAChR or any other neuroreceptor is still missing. If available, it could provide a molecular correlate for the recognition function of the receptor and thereby also for rational drug design.Prompted by the fact that all attempts to crystallize the detergent-solubilized whole nAChR protein have been unsuccessful for the past more than 20 years, we have begun to overexpress selected domains of the receptor in bacterial expression systems. If successfully renatured, the domains that are not transmembranous should be water-soluble and thus should provide a better material for protein crystallization than the detergent-solubilized whole receptor protein. Moreover, if small enough in size, such fragments should be suited for multidimensional NMR analysis (13). As presented here for the N-terminal extracellular region of the nAChR ␣-subunit, we attempted and achieved expression as a water-soluble globular protein that displays ligand binding properties comparable to or better than those of the SDS gel-isolated ␣-subunit. To achieve appropriate renaturation, we developed a protocol for the complete unfolding (by means of chaotropic agents and disulfide-reducing agents) and refolding (by means of an oxido shuffling system and L-arginine as structure-stabilizing agent) of the expressed polypeptide (14). The experimental conditions were such that self-organization of the unfolded protein was favored at the expense of (unwanted) aggregation and denaturation. In this way, we are able to prepare large quantities of the functional ligand binding domain from inclusion bodies of transformed Escherichia coli bacteria. Our results confirm that the N-terminal extracellular domain indeed harbors major elements of the ligand recognition function of the nAChR, as has long been suggested on the basis of affinity labeling and immunological studies (for recent reviews, see Refs. 1,2,8,9,15,and 16). EXPERIMENTAL PROCEDURE...

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“…The nicotinic acetylcholine receptor (nAChR) 1 from muscle is a pentamer of homologous subunits surrounding a central channel with stoichiometries of ␣ 2 ␤␥␦ (embryonic muscle) or ␣ 2 ␤⑀␦ (adult muscle) (1)(2)(3)(4). The amino-terminal 210 amino acids principally form the extracellular domain that harbors the agonist binding sites.…”

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confidence: 99%

Pairwise Electrostatic Interactions between α-Neurotoxins and γ, δ, and ε Subunits of the Nicotinic Acetylcholine Receptor

Osaka¹,

Malany²,

Molles³

et al. 2000

Journal of Biological Chemistry

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␣-Neurotoxins bind with high affinity to ␣-␥ and ␣-␦ subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of ϳ1200 Å 2 and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound ␣-neurotoxin. couples minimally to charged ␣-toxin residues. Arg 36 , despite strong energetic contributions, does not partner with any ␥ subunit residues, perhaps indicating its proximity to the ␣ subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at ␥176 and ␥119 can be distinguished from those at ␥55. The nicotinic acetylcholine receptor (nAChR)1 from muscle is a pentamer of homologous subunits surrounding a central channel with stoichiometries of ␣ 2 ␤␥␦ (embryonic muscle) or ␣ 2 ␤⑀␦ (adult muscle) (1-4). The amino-terminal 210 amino acids principally form the extracellular domain that harbors the agonist binding sites. Agonists and competitive antagonists occupy binding sites formed at two of the five subunit interfaces, ␣␥ (or ⑀) and ␣␦. Simultaneous occupation of both interfaces by agonist is required for channel opening, whereas occupation of a single site by antagonist is sufficient to antagonize an agonist response (5, 6). Chang and Lee (8) observed that ␣-bungarotoxin, a member of the ␣-neurotoxin family, irreversibly blocked the responses to acetylcholine in muscle. Changeux and colleagues (9) subsequently employed this toxin to provide the first characterization of a pharmacological receptor. Since then, the ␣-neurotoxins have played a pivotal role in localization, identification, and purification of nicotinic receptors. Although over 100 short (60 -62 amino acids) and long (66 -74 amino acids) ␣-neurotoxins have been isolated, sequenced, and characterized, little is known about the basis of their high affinity and their exquisite sensitivity for certain subtypes of nicotinic receptors. The ␣-neurotoxins share a common basic structure consisting of three large polypeptide loops emerging from a smaller globular core (10). X-ray crystallographic studies of a structurally homologous toxin, fasciculin, bound to its target acetylcholinesterase, reveal a high degree of complementarity between the surfaces and an interfacial contact zone extending over 1200 Å and encompassing nearly 30% of the ␣-toxin surface (11,12).In previous investigations of ␣-neurotoxin-nAChR interactions, we found that the binding site formed at the ␣⑀ interface shows unique resistance to binding Naja mossambica mossambica I (NmmI) toxin due to the presence of Thr 176 and Ala 177 in the ⑀ subunit. Furthermore, substitution of ⑀ subunit residues at homologous positions in the ␥ subunit demonstrated a strong linkage between Lys 27 on the second loop of NmmI ␣-toxin and Glu 176 on the ␥ subunit of the receptor (13). We have extended this study to examine ␥ subunit contributions to NmmI binding and find Trp 55 , Leu 119 , and Asp 174 on the ␥ subunit to also res...

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The Emerging Three‐Dimensional Structure of a Receptor

Cited by 203 publications

References 187 publications

Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Lateral Diffusion, Function, and Expression of the Slow Channel Congenital Myasthenia Syndrome αC418W Nicotinic Receptor Mutation with Changes in Lipid Raft Components

Expression and Renaturation of the N-terminal Extracellular Domain of Torpedo Nicotinic Acetylcholine Receptor α-Subunit

Pairwise Electrostatic Interactions between α-Neurotoxins and γ, δ, and ε Subunits of the Nicotinic Acetylcholine Receptor

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