Relative Spatial Position of a Snake Neurotoxin and the Reduced Disulfide Bond α(Cys192-Cys193) at the αγ Interface of the Nicotinic Acetylcholine Receptor

Michalet, Sophie; Teixeira, Fátima C.; Gilquin, Bernard; Mourier, Gilles; Servent, Denis; Drevet, Pascal; Binder, Patrice; Tzartos, Socrates J.; Ménèz, Andre; Kessler, Pascal

doi:10.1074/jbc.m002362200

Cited by 17 publications

(32 citation statements)

References 48 publications

(75 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Several Cys-substituted mutants of Naja nigricollis ␣-neurotoxin, a short-chain neurotoxin, were cross-linked to the Torpedo ␣ subunit with various efficiencies depending on the spacer length of the dimaleimide derivative used and the site of toxin mutagenesis. It was concluded that Cys 192 and Cys 193 were located under the tip of the first loop, ϳ11.5 Å from the ␣-carbon at toxin position 10, and close to the second loop, ϳ 15.5 Å from the ␣-carbon at toxin position 33 (51). This orientation is entirely consistent with the structure shown in Fig.…”

Section: Preparation Ofsupporting

confidence: 77%

The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica

Zeng

Moise

Grant

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

The region encompassing residues 181-98 on the ␣1 subunit of the muscle-type nicotinic acetylcholine receptor forms a major determinant for the binding of ␣-neurotoxins. We have prepared an 15 N-enriched 18-amino acid peptide corresponding to the sequence in this region to facilitate structural elucidation by multidimensional NMR. Our aim was to determine the structural basis for the high affinity, stoichiometric complex formed between this cognate peptide and ␣-bungarotoxin, a long ␣-neurotoxin. Resonances in the complex were assigned through heteronuclear and homonuclear NMR experiments, and the resulting interproton distance constraints were used to generate ensemble structures of the complex. The nicotinic acetylcholine receptor (nAChR) 1 (1) has long been a prototype for ligand-gated ion channels. This receptor is involved in excitatory synaptic transmission at the neuromuscular junction and also plays an important role in the nervous system. The nAChRs are pentameric complexes composed of homologous subunits with subunits arranged around the central channel in a symmetrical manner. The muscle-type nAChR contains two ␣1 subunits and one each of the ␤1, ␥(⑀), and ␦ subunits. The ligand binding sites are situated at the ␣␥(⑀) and ␣␦ subunit interfaces. The muscle-type nAChR serves as an important model for the study of the structures and functions of related ligand-gated ion channels (for review, see Ref.

show abstract

Section: Preparation Ofsupporting

confidence: 77%

The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica

Zeng

Moise

Grant

et al. 2001

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…[48][49][50][51] The ra-CbT is chemically, functionally and structurally indistinguishable from the venom toxin and is produced with a yield of approximately 1.2 mg/L of culture. 48,49 The use of ra-CbT avoids any problems of conflicting results because of contamination of ''purified'' drug with residual that can have very similar molecular mass, as in the case of j-BTX in ''purified'' a-BTX.…”

Section: Specificity Of A-cbt In Inhibiting Tumor Developmentmentioning

confidence: 99%

Retracted: Inhibition of non‐neuronal α7‐nicotinic receptor reduces tumorigenicity in A549 NSCLC xenografts

Paleari

Sessa

Catassi

et al. 2009

Intl Journal of Cancer

Self Cite

View full text Add to dashboard Cite

Nicotinic acetylcholine receptors (nAChR) are expressed on bronchial epithelial and non-small cell lung cancer cells and are involved in cell growth regulation. Nicotine (classical nAChR agonist) induced cell proliferation, whereas nAChR antagonists, dtubocurarine or a-cobratoxin (a-CbT), induced cell death. In the current study, we further explored the antitumor potential mechanisms and activities of a-CbT. NOD/SCID mice were grafted intraperitoneally or orthotopically and treated with a-CbT. a-CbT treatment [0.04 ng/kg or 0.12 ng/kg] induced a strong reduction in tumor size (90%) in comparison with mice treated with the vehicle alone. Tumor inhibition was related to severe induction of apoptosis. Moreover, neoangiogenesis was strongly inhibited (reduction of cells positive to vascular endothelial growth factor and CD31). Biochemical analyses of the cells, isolated by the primary lung tumor in a-CbT-treated mice, showed apoptosis features characterized by: (i) inhibition of BAD phosphorylation at Ser 112 and Ser 136; (ii) BAD dissociation from 14-3-3; (iii) BAD association with BCL-XL; and (iv) cleavage of caspase-9. Moreover, these cells were unable to grow in soft agar and develop tumor, when reinjected into mice. The small interfering RNA-mediated silencing of the a7-nAChR gene confirmed that a-CbT specifically inhibited the a7-nAChR-mediated survival pathway in A549 cells. Furthermore, the specificity of a-CbT is reinforced by the lack of effect of short chain toxin (Erabutoxin-a). Once more, the no effect of the low-affinity R33E-modified a-CbT strengthened the specificity of this inhibition. Although a7-nAChR antagonists, such as a-CbT, are unlikely to be a primary therapy, it may provide lead compounds for the design of clinically useful drugs. ' UICCKey words: nicotinic acetylcholine receptors; cobratoxin; mouse model lung cancer; tumor-induced apoptosis; neoangiogenesis inhibition Lung cancer is the leading cause of cancer mortality worldwide. Epidemiologic studies have demonstrated that cigarette smoking is the major risk factor in its development, with a striking doseresponse relationship. It is estimated that 80-90% of lung cancer incidence can be attributed to cigarette smoking.1 Despite years of research, the prognosis for patients with lung cancer remains dismal, with a 5-year survival rate of 14%. Currently, the usual chemotherapy regimen (adjuvant or neoadjuvant) combines a platinum-containing drug with another cytotoxic agent (i.e., gemcitabin, Taxol or navelbine). However, platinum-based therapies have drawbacks: severe toxic effects for the patient and drug resistance in the tumor cells.2 Future directions for treatment are heavily weighted toward targeted therapies, namely those aimed at molecular abnormalities involved in the pathogenesis of lung cancer 3 rather than traditional cytotoxic agents. 4 Currently, it has been established that acetylcholine (ACh) is widely synthesized by a variety of non-neuronal cell types, including airway epithelial cells. [5][6][7][8][9][10][11][12][13][14...

show abstract

“…Clearly the loop C that contains highly functional residues conserved in both receptors plays a predominant binding role. The evidence that supports that conclusion includes (i) mutational analyses with short chain (33,34) and long chain (35)(36)(37) toxins, (ii) the use of synthetic receptor peptides of the region 180-200 (38,39), (iii) studies on the resistance of various species to toxins (40,41), (iv) direct affinity labeling experiments (42), and (v) resolution of solution structures of the complexes formed between ␣-Bgtx and peptides (32,(43)(44)(45). Also, the additional binding function observed here for other ␣ and non-␣ loops was postulated for muscular receptors (46)(47)(48).…”

Section: Discussionmentioning

confidence: 99%

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

Fruchart-Gaillard

Gilquin

Antil-Delbeke

et al. 2002

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

Self Cite

120

150

View full text Add to dashboard Cite

To understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the ␣-cobratoxin-␣7 receptor complex. This model was achieved by using (i) a three-dimensional model of the ␣7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the ␣-cobratoxin and ␣7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. T he ␣-neurotoxins from snake venom are potent antagonists that block nicotinic acetylcholine receptors (AChRs) and hence affect synaptic transmission (1-3). Despite many studies (reviewed in ref. 4), the molecular process associated with this efficient blockage remains unclear. To approach this question, we previously studied ␣-cobratoxin (␣-Cbtx), an ␣͞K neurotoxin that binds to both muscular and homopentameric neuronal receptors (␣7 and ␣8) with high affinities (4). This toxin, similar to other snake neurotoxins, is folded into three adjacent loops rich in ␤-sheet that emerge from a small globular core in which four disulfide bonds are located (5). By mutational analyses, the residues by which ␣-Cbtx interacts with the muscular-type or neuronal ␣7 receptors were identified previously (6, 7). The present study shows how functional residues account for the antagonistic properties of the toxin toward the ␣7 neuronal receptor. The ␣7 AChR possesses five identical ␣7 subunits (8) that offer five ligand-binding sites located at the interface of two subunits (9). These sites include residues located on the different functional loops described previously on the principal ␣7 (ϩ) face, loops A, B, and C and on the complementary ␣7 (Ϫ) face, loops D, E, and F (refs. 10-13; see Fig. 1). Until now, the residues of the ␣7 receptor involved in snake toxin binding have remained unknown.The aim of the present paper is fourfold. First, by an extensive mutational study we have identified ␣7 receptor residues involved in the interaction with ␣-Cbtx. Second, by using a double-mutant cycle approach we have disclosed several pairs of interacting residues in the toxin-receptor complex. Third, by using the three-dimensional (3D) structure of an AChBP that is similar functionally and structurally to the N-terminal domain of an AChR ␣-subunit (14), we used a 3D model for the ␣7 subunit extracellular region obtained by comparative modeling [see accompanying paper on page 3210 (15)]. Fourth, by using this model, a molecular dynamics simulation of the loop F region, and the constraints derived from our pairwise analysis, we propose an experimentally based 3D model of the complex between the ␣-Cbtx and ␣7 receptor, which explains the antagonistic properties of the snake toxin toward the neuronal recepto...

show abstract

Relative Spatial Position of a Snake Neurotoxin and the Reduced Disulfide Bond α(Cys192-Cys193) at the αγ Interface of the Nicotinic Acetylcholine Receptor

Cited by 17 publications

References 48 publications

The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica

The Solution Structure of the Complex Formed between α-Bungarotoxin and an 18-mer Cognate Peptide Derived from the α1 Subunit of the Nicotinic Acetylcholine Receptor from Torpedo californica

Retracted: Inhibition of non‐neuronal α7‐nicotinic receptor reduces tumorigenicity in A549 NSCLC xenografts

Experimentally based model of a complex between a snake toxin and the α7 nicotinic receptor

Contact Info

Product

Resources

About