Three‐dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution

Arnoux, B.; Ménez, R.; Drevet, Pascal; Boulain, Jean‐Claude; Ducruix, A.; Ménèz, Andre

doi:10.1016/0014-5793(94)80574-1

Cited by 13 publications

(8 citation statements)

References 11 publications

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“…28 and 29), which form high affinity complexes with the receptor; for example, the ␣-bungarotoxin-receptor complex has a K d of ϳ10 Ϫ12 . The structure of the ␣-neurotoxins has been solved through nuclear magnetic resonance (30 -32) and x-ray crystallographic studies (33)(34)(35). These polypeptides (ϳ7 kDa) are characterized by three large loops which extend from a rigid globular domain held together by 4 or 5 conserved disulfide bonds.…”

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confidence: 99%

Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

Ackermann

Ang

Kanter

et al. 1998

Journal of Biological Chemistry

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␣-Neurotoxins are potent inhibitors of the nicotinic acetylcholine receptor (nAChR), binding with high affinity to the two agonist sites located on the extracellular domain. Previous site-directed mutagenesis had identified three residues on the ␣-neurotoxin from Naja mossambica mossambica ( The nicotinic acetylcholine receptor (nAChR) 1 is a member of the large family of neurotransmitter-gated ion channels (for review, see Refs. 1-4). It is composed of five subunits that are arranged in the circular order of ␣␥␣␦␤. The two ligand binding sites reside in the extracellular domain at the ␣␥ and ␣␦ subunit interfaces (5, 6). A high resolution structure is not available for any member of the family of neurotransmitter-gated ion channels. However, within this family the structure of the nAChR has been most extensively characterized through sitedirected labeling (7-12), site-specific mutagenesis (13-23), electron microscopy reconstruction analysis (24, 25), and homology modeling (26). In current models, three discontinuous regions or domains on the ␣-subunit (encompassing residues around 93, 149 -154, and 180 -200) and four regions on the ␥/␦ subunits are thought to participate in the formation of the binding sites (2, 26). Because of sequence differences in the ␦ and ␥ subunits, the binding sites on the receptor are not identical; consequently, many ligands bind to each site with different affinities (15,22,27).The critical tool utilized in the initial identification of the receptor and in subsequent structural analyses is the family of three-fingered snake ␣-neurotoxins (for review, see Refs. 28 and 29), which form high affinity complexes with the receptor; for example, the ␣-bungarotoxin-receptor complex has a K d of ϳ10Ϫ12 . The structure of the ␣-neurotoxins has been solved through nuclear magnetic resonance (30 -32) and x-ray crystallographic studies (33-35). These polypeptides (ϳ7 kDa) are characterized by three large loops which extend from a rigid globular domain held together by 4 or 5 conserved disulfide bonds. Even though ␣-neurotoxin structures have been solved, little is known about the structure of the toxin-receptor complex, and interacting residues have not been identified.In previous work, we delineated residues involved in the binding interaction on both the ␣-neurotoxin, Naja mossambica mossambica (NmmI), and the mouse muscle nAChR interfaces (36). Four residues located on the receptor ␣-subunit and three residues located on the toxin structure were found to contribute significantly to high affinity binding. Even though wild-type NmmI displays an equivalent affinity for the two binding sites on nAChR, we showed through mutational analysis that the energetic contribution of selected residues differed at the two sites. Several of the toxin and receptor mutations studied differentially affected binding at the ␣␦ and ␣␥ sites, resulting in two distinct binding affinities (36). In this study, we have utilized double mutant cycles to explore whether any of these residues are involved in pairwise interact...

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confidence: 99%

Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

Ackermann

Ang

Kanter

et al. 1998

Journal of Biological Chemistry

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“…As previously reported (74,77), the polypeptide chain of Ea is organized into three adjacent loops forming a large ␤-pleated sheet, which emerges from a small globular core where are located the 4 disulfides. The toxin has two opposite faces (Fig.…”

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confidence: 78%

“…03 --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- its functional (30) and structural properties (74). Using the same strategy, as many as 51 Ea mutants have been thus prepared.…”

Section: Fig 4 Inhibition Of Binding Of 3 H-labeled Toxin To M␣2-3 mentioning

confidence: 99%

Mimicry between Receptors and Antibodies

Ducancel

Mérienne

Fromen-Romano

et al. 1996

Journal of Biological Chemistry

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In several instances, a monoclonal antibody raised against a receptor ligand has been claimed to mimic the ligand receptor. Thus, a specific monoclonal antibody (M␣2-3) raised against a short-chain toxin from snake was proposed to mimic the nicotinic acetylcholine receptor (AChR) (1). Further confirming this mimicry, we show that (i) like AChR, M␣2-3 elicits anti-AChR antibodies, which in turn elicit anti-toxin antibodies; and (ii) the region 106 -122 of the ␣-chain of AChR shares 66% primary structure identity with complementarity-determining regions of M␣2-3. Also, a mutational analysis of erabutoxin a reveals that the epitope recognized by M␣2-3 consists of 10 residues, distributed within the three toxin loops. Eight of these residues also belong to the 10-residue epitope recognized by AChR, a result that offers an explanation as to the functional similarities between the receptor and the antibody. Strikingly, however, most of the residues common to the two epitopes contribute differentially to the energetic formation of the antibody-toxin and the receptor-toxin complexes. Together, the data suggest that the mimicry between AChR and M␣2-3 is partial only.

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“…With the view to identify the functional site of Ea, its cDNA has been previously cloned and expressed in Escherichia coli as a fusion protein [14], which was then cleaved [15]. The resulting recombinant toxin was indistinguishable from the snake toxin regarding both its biological activity [15] and its three‐dimensional structure [16]. Using this expression system, a large panel of Ea mutants has been produced and used to identify the residues by which the toxin binds to both AchR [5,6] and an AchR‐mimicking antibody [17].…”

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confidence: 99%

“…Ea S8G. The orientation and location of the Ea S8G mutant molecule were checked by rigid‐body refinement at 0.3 nm resolution, using coordinates from the recombinant Ea structure [16]. Refinement was done using the x plor program [23].…”

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High resolution X‐ray analysis of two mutants of a curaremimetic snake toxin

Gaucher

Ménez

Arnoux

et al. 2000

European Journal of Biochemistry

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A previous mutational analysis of erabutoxin a (Ea), a curaremimetic toxin from sea snake venom, showed that the substitutions S8G and S8T caused, respectively, 176-fold and 780-fold affinity decreases for the nicotinic acetylcholine receptor (AchR). In view of the fact that the side-chain of Ser8 is buried in the wild-type toxin, we wondered whether these affinity changes reflect a direct binding contribution of S8 to the receptor and/or conformational changes that could have occurred in Ea as a result of the introduced mutations. To approach this question, we solved X-ray structures of the two mutants S8G and S8T at high resolution (0.18 nm and 0.17 nm, with R factors of 18.0% and 17.9%, respectively). The data show that none of the mutations significantly modified the toxin structure. Even within the site where the toxin binds to the receptor the backbone conformation remained unchanged. Therefore, the low affinities of the mutants S8T and S8G cannot be explained by a large conformational change of the toxin structure. Although we cannot exclude the possibility that undetectable structural changes have occurred in the toxin mutants, our data support the view that, although buried between loop I and II, S8 is part of the functional epitope of the toxin.Keywords: curaremimetic toxins; nicotinic acetylcholine receptor; X-ray structure.Mutational analyses are widely used to identify the functional surfaces of proteins and to determine the contribution of interacting residues to the energetic stabilization of protein± protein complexes [1,2]. In most cases, it is assumed that the introduced mutations have little, if any, effect on the protein conformation, leading authors to conclude that affinity decreases occurring as a result of mutations are likely to reflect a direct contribution of the mutated residue to protein interaction [3]. In general, this conclusion appears to be correct, especially when it is supported by crystallographic data that indicate that the mutated residue establishes contacts with the interacting partner [3,4]. However, the situation may be more complex, as some mutations cause affinity decreases even though the mutated residue is not in the region contacting the receptor [4]. Therefore, in the absence of information regarding the three-dimensional structure of a protein±protein complex, the effect of some mutations should be considered with caution, the introduced mutation causing possibly local or even more global conformational changes in the protein. One way to tentatively clarify a doubtful case consists of elucidating the three-dimensional structure of the mutant and comparing it with that of the wild-type protein. This situation has been met with mutations introduced at position 8 of erabutoxin a (Ea), a snake curaremimetic toxin, the functional site of which has been identified on the basis of extensive mutational analyses [5,6].Ea is a small protein of 62 amino acids that belongs to the family of short-chain curaremimetic toxins [7], which bind to the nicotinic acetylcholine recept...

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Three‐dimensional crystal structure of recombinant erabutoxin a at 2.0 Å resolution

Cited by 13 publications

References 11 publications

Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

Identification of Pairwise Interactions in the α-Neurotoxin-Nicotinic Acetylcholine Receptor Complex through Double Mutant Cycles

Mimicry between Receptors and Antibodies

High resolution X‐ray analysis of two mutants of a curaremimetic snake toxin

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