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...

Venoms of elapid and hydrophid snakes contain a family of small toxic proteins called curarimimetic toxins or ␣-neurotoxins that bind with high affinity to muscular nicotinic acetylcholine receptors (AChRs) 1 and hence affect synaptic transmission (1, 2). All these toxins adopt a leaf-like shape with three adjacent loops rich in ␤-sheet that emerge from a small globular core where four disulfide bonds are invariably located (3-7). Notwithstanding their common fold and their similar biological function, ␣-neurotoxins are currently classified as short chain toxins with 60 -62 residues and four disulfide bonds and long chain toxins with 66 -74 residues and five disulfide bonds. In agreement with this old chemically based classification, we recently showed that the long chain toxins are also and uniquely capable of binding with high affinity to the neuronal ␣7 receptor (8). These preliminary data also indicated that the neuron-specific binding capacity may be associated with the unique presence in the long chain toxins of a small cyclic loop at the tip of their central loop. The goal of this work was therefore to identify as precisely as possible the determinants by which long chain toxins bind to the neuronal ␣7-AChR and to compare them with those involved when toxins bind to the muscular AChR.The toxin used in this study is ␣-cobratoxin (␣-Cbtx) (9) from Naja naja siamensis (probably Naja kaouthia (10)). It is a prototype of long chain curarimimetic toxins with a single polypeptide chain of 71 amino acids and five disulfide bonds. ␣-Cbtx binds with high affinity to the muscular type AChR from Torpedo marmorata (K d ϭ 58 pM) and the neuronal ␣7-AChR (K d ϭ 9 nM). Its three-dimensional structure is known from both NMR (11) and x-ray crystallographic studies (12). We recently submitted this toxin to an extensive site-directed mutagenesis and found that the residues by which it binds to the Torpedo AChR include a number of amino acids that are highly conserved throughout the family of curarimimetic toxins (13). These are Lys-23, Trp-25, Asp-27, Phe-29, and Arg-33, which belong to the concave face of the toxin loop II, and Lys-49, which belongs to the same face of loop III. The same residues of a short chain curarimimetic toxin are involved in binding to the same AChR (14,15). In addition, however, long and short chain curarimimetic toxins use specific residues for binding to the Torpedo AChR. These specific residues are located in the Cterminal tail and in loop I of long and short chain toxins, respectively (13).The goal of this study was 3-fold. First, using a set of 36 toxin mutants, we identified the residues by which ␣-Cbtx most likely binds to the neuronal ␣7 receptor. Second, we compared these data with those that previously indicated the residues by which the same toxin binds to the Torpedo AChR (13). Third, to identify the regions of the ␣7 receptor that are recognized by the toxin, we mutated different residues in various functional loops of the ␣7 receptor and studied the effect of these muta-* The costs of ...

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