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

Abstract

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...

Structural and Functional Characterization of a Novel Homodimeric Three-finger Neurotoxin from the Venom of Ophiophagus hannah (King Cobra)

“…Functionally Important Residues for Haditoxin-Extensive structure-function relationship studies on the short-chain ␣-neurotoxin, erabutoxin-a (24,65,66), and the long-chain ␣-neurotoxins, ␣-cobratoxin (67, 68) and ␣-bungarotoxin (69,70), revealed the critical residues involved in the recognition of nAChRs by snake neurotoxins. The crucial residues for ␣-neurotoxins to bind to muscle (␣␤␥␦) nAChRs are Lys-27, Trp-29, Asp-31, Phe-32, Arg-33, and Lys-47.…”

Models of the extracellular domain of the nicotinic receptors and of agonist- and Ca 2+ -binding sites

“…Accordingly, the toxin could recognize neither the conformation of AChBP that has been crystallized nor our own models of nAChRs, presented in the present article, but rather another conformation carrying a more opened agonist- binding site. The accompanying paper by Fruchart-Gaillard et al (10) presents a model of ␣7 with an agonist-binding site modified to accommodate the ␣-cobratoxin. When we dock the small ligands on this model, we get significantly lower affinities, 100-fold for epibatidine and 10-fold for nicotine and acetylcholine.…”

Structural Model of Ligand-G Protein-coupled Receptor (GPCR) Complex Based on Experimental Double Mutant Cycle Data

“…Ambiguous distance restraints taking into account this uncertainty were used. Docking of MT7 onto hM1 dimer was basically performed following the same protocol that we and other authors used previously (41)(42)(43). The protocol was adapted to three partners: MT7 and the two hM1 protomers (see "Experimental Procedures").…”