Direct structural localization of two toxin-recognition sites on an ACh receptor protein

Zingsheim, H. P.; Barrantes, Francisco J.; Frank, Joachim; Hänicke, W.; Neugebauer, D.-Ch.

doi:10.1038/299081a0

Cited by 98 publications

(33 citation statements)

References 18 publications

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“…However, we cannot exclude the possibility of only partial occupancy of antibody in the available RyR1 binding sites because of the binding conditions and interference of the detergent at a relatively high concentration (ϳ0.4% CHAPS). A statistical analysis of the difference map shows that the regions with the highest t values (significance level Ͼ98%) are located within the areas of highest positive densities in the difference map, reinforcing their statistical significance (23)(24)(25). Two other smaller regions of positive differences are detected in the central portion of the channel.…”

Section: Resultsmentioning

confidence: 98%

See 1 more Smart Citation

The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain

Baker

Serysheva

Sencer

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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We used a combination of bioinformatics, electron cryomicroscopy, and biochemical techniques to identify an oxidoreductase-like domain in the skeletal muscle Ca 2؉ release channel protein (RyR1). The initial prediction was derived from sequence-based fold recognition for the N-terminal region (41-420) of RyR1. The putative domain was computationally localized to the clamp domain in the cytoplasmic region of a 22Å structure of RyR1. This localization was subsequently confirmed by difference imaging with a sequence specific antibody. Consistent with the prediction of an oxidoreductase domain, RyR1 binds [ 3 H]NAD ؉ , supporting a model in which RyR1 has a oxidoreductase-like domain that could function as a type of redox sensor. During excitation-contraction coupling in skeletal muscle, Ca 2ϩ is released from the lumen of the sarcoplasmic reticulum (SR) via the Ca 2ϩ release channel, also known as the ryanodine receptor, RyR1. In skeletal muscle, the Ca 2ϩ release channel is physically coupled to the L-type voltage dependent Ca 2ϩ channel dihydropyridine receptor (DHPR), such that a depolarization induced change in the conformation of DHPR induces the opening of ryanodine receptor 1 (RyR1). This leads to an increase of cytoplasmic Ca 2ϩ , triggering a sequence of events that lead to muscle contraction. RyR1 is a homotetramer (1) whose subunits are ϳ565 kDa (e.g., human, 5,038 residues; rabbit, 5,037 residues) (2, 3). Mutations in three domains of this protein, one of which is between amino acids 35 and 614, have been implicated in the pathogenesis of two human diseases, malignant hyperthermia and central core disease (4, 5).The Ca 2ϩ release channel exists in at least two functional states, opened and closed (6), which likely have conformational differences. The low-resolution structures of the Ca 2ϩ release channel in different functional states have been studied extensively by electron cryomicroscopy (7-9). On opening, a number of structural changes occur in several regions of the channel, including both the clamplike domains in the cytoplasmic region and the transmembrane domain. The clamp domains are the most likely candidates for interaction with DHPR (8) and must, therefore, be allosterically coupled to the transmembrane domain in order for DHPR to induce the opening of the Ca 2ϩ permeable pore of RyR1. Here we describe a unique approach for identification of new functional and structural domains of this complex protein. MethodsSequence Analysis. Initial motif searching in the primary sequence of rabbit RyR1 (P11716) was done by using PROSCAN (10) with a threshold of 70%. Subsequently, 500-residue consecutive, serial sequence segments of the RyR1 were submitted to the University of California, Los Angeles-Department of Energy (UCLA-DOE) Fold recognition server (11). Primary sequence alignments were performed by using CLUSTALW (Gonnet weight matrix) with a Gonnet Pam250 positive-value similarities scoring system (12, 13). Additionally, multiple sequence alignments were done with other RyR sequences. As sequenc...

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Section: Resultsmentioning

confidence: 98%

“…The differences were considered to be significant at the confidence level greater than 98% (i.e., random chance is Ͻ0.02) (23)(24)(25). and k obs is the slope obtained from a plot of ln(B e ͞(B e Ϫ B t )) where B e is the amount bound at equilibrium and B t is the amount bound at time t.…”

Section: Methodsmentioning

confidence: 99%

The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain

Baker

Serysheva

Sencer

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Individual subunits appeared as barrel staves surrounding the vestubule, and differences in electron densities within each subunit could be discerned. Assignment of subunit location was tentatively achieved using ␣-bungarotoxin and subunit-specific antibodies resolvable as new densities in the electron density maps (Zingsheim et al, 1982;Kubalek et al, 1987). From these studies, the two ␣-subunits were shown to be separated by another subunit, apparently the ␤-subunit, and the ␦-subunit, which covalently joined pairs of receptors, was shown not to be the subunit between the two ␣-subunits (reviewed by Karlin, 1987).…”

Section: Early Advances (1980s Through 1990s)mentioning

confidence: 99%

The nicotinic receptor ligand binding domain

2002

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ABSTRACT:The ligand binding domain (LBD) of the nicotinic acetylcholine receptor has served as a prototype for understanding molecular recognition in the family of neurotransmitter-gated ion channels. During the past fifty years, studies progressed from fundamental electrophysiological analyses of ACh-evoked ion flow, to biochemical purification of the receptor protein, pharmacological measurements of ligand binding, molecular cloning of receptor subunits, site-directed mutagenesis combined with functional analysis and recently, atomic structural determination. The emerging picture of the nicotinic receptor LBD is a specialized pocket of aromatic and hydrophobic residues formed at interfaces between protein subunits that changes conformation to convert agonist binding into gating of an intrinsic ion channel.

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“…The evidence in support of this assertion is twofold : the high surface density of binding sites for the quasi-irreversible competitive antagonist a-bungarotoxin, and the equivalent coherent coverage of the membrane by the 7 -8-nm particles attributed to the receptor protein (see reviews in [1,2] and references therein). In spite of this high density and close packing of the receptor molecules, naturally occurring ordered arrays are rare (0.1 -1 in receptor-rich membranes [3]). The hexagonal outline observed in some electron micrographs of the receptor particles is a result of the local tight packing rather than a consequence, or reflection, of any internal symmetry feature [3].…”

mentioning

confidence: 99%

“…In spite of this high density and close packing of the receptor molecules, naturally occurring ordered arrays are rare (0.1 -1 in receptor-rich membranes [3]). The hexagonal outline observed in some electron micrographs of the receptor particles is a result of the local tight packing rather than a consequence, or reflection, of any internal symmetry feature [3]. This lack of crystalline order does not necessarily imply a random organization of the receptor in the membrane, and in fact we have shown [4] that the acetylcholine receptor particles do possess the ability to form oligomeric aggregates, in agreement with the observation of linear double or quadruple structures by freezeetching electron microscopy [5].…”

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confidence: 99%

Rotational Molecular Dynamics of the Membrane-Bound Acetylcholine Receptor Revealed by Phosphorescene Spectroscopy

1981

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The rotational mobility of the membrane-bound acetylcholine receptor from Torpedo marmorata has been studied by phosphorescence anisotropy techniques using eosin-5'-isothiocyanate and eosin-5'-iodoacetamide derivatives of the a-neurotoxin of Bungarus multicinctus (a-bungarotoxin). Normal membranes, those depleted of nonreceptor peripheral peptides by alkaline treatment, as well as membranes subjected to various chemical modifications of the thiol groups, have been characterized. Rotational correlation times (10 -26 ps) compatible with the motion of individual 9-S monomeric receptor species of M , 250000 were observed upon reduction of the membranes with dithiothreitol or by raising the temperature to 39 "C in the case of alkaline-treated membranes. Membranes prepared throughout in N-ethylmaleimide, which yield upon detergent solubilization a predominant 13-S diiiieric species and which are not depleted of the nonreceptor v-peptide ( M , 43000) by alkaline treatment, are relatively more 'rigid' than normal membranes and do not show the anisotropy components characteristic of the monomeric receptor. The influence of the v-peptide on the structural stability of the receptor oligomeric forms and the thiol-dependent interconversions are thus reflected in the time-dependent spectroscopic measurements.In the adult cholinergic synapse, the post-synaptic membrane is densely covered with acetylcholine receptor molecules. The evidence in support of this assertion is twofold : the high surface density of binding sites for the quasi-irreversible competitive antagonist a-bungarotoxin, and the equivalent coherent coverage of the membrane by the 7 -8-nm particles attributed to the receptor protein (see reviews in [1,2] and references therein). In spite of this high density and close packing of the receptor molecules, naturally occurring ordered arrays are rare (0.1 -1 in receptor-rich membranes [3]). The hexagonal outline observed in some electron micrographs of the receptor particles is a result of the local tight packing rather than a consequence, or reflection, of any internal symmetry feature [3]. This lack of crystalline order does not necessarily imply a random organization of the receptor in the membrane, and in fact we have shown [4] that the acetylcholine receptor particles do possess the ability to form oligomeric aggregates, in agreement with the observation of linear double or quadruple structures by freezeetching electron microscopy [5].The high specialized post-synaptic membrane in the cholinergic synapse differs significantly from other biological membranes. In fact, the usual view of the latter as a matrix of lipid interrupted by integral membrane proteins contemplated by the so-called 'fluid mosaic model ' [6], would certainly fail to serve as an adequate archetype of the membrane in question. Instead, the rather heretic view of a protein matrix with lipids filling the interstices between the individual protein components is suggested by the available evidence (see [I] and references therein) though heed is no...

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Direct structural localization of two toxin-recognition sites on an ACh receptor protein

Cited by 98 publications

References 18 publications

The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain

The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain

The nicotinic receptor ligand binding domain

Rotational Molecular Dynamics of the Membrane-Bound Acetylcholine Receptor Revealed by Phosphorescene Spectroscopy

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Direct structural localization of two toxin-recognition sites on an ACh receptor protein

Cited by 98 publications

References 18 publications

The skeletal muscle Ca 2+ release channel has an oxidoreductase-like domain

The skeletal muscle Ca 2+ release channel has an oxidoreductase-like domain

The nicotinic receptor ligand binding domain

Rotational Molecular Dynamics of the Membrane-Bound Acetylcholine Receptor Revealed by Phosphorescene Spectroscopy

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The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain

The skeletal muscle Ca ²⁺ release channel has an oxidoreductase-like domain