Site‐directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

The aim of this study was to present a new concept of site‐directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 μ M for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys‐192 and Cys‐193 on the α ‐subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site‐directed reducing agent (ARA) than with DTT, and was highly specific for the α ‐subunits. The contribution of the carbamylcholine moiety of the site‐directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 μ M), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 μ M of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site‐directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site

Variability among the Sites by Which Curaremimetic Toxins Bind to Torpedo Acetylcholine Receptor, as Revealed by Identification of the Functional Residues of α-Cobratoxin

International audienc

Rôle des toxines dans l'étude fonctionnelle et structurale des récepteurs nicotiniques de l'acétylcholine

Les toxines animales ont souvent été très utiles dans la compréhension des modes de fonctionnement de leurs différentes cibles. Celles qui interagissent au niveau des récepteurs nicotiniques de l'acétylcholine sont purifiées à partir de venin de serpents ou de cônes marins. Pour leur haute affinité et leur sélectivité d'interaction, ces toxines sont utilisées depuis une trentaine d'années comme de véritables sondes moléculaires permettant d'identifier, de localiser et de purifier ces récepteurs. Par ailleurs, ces toxines ont joué un rôle majeur dans la compréhension des spécificités fonctionnelles des différents sous-types de récepteurs et ont permis d'accéder à des informations structurales les concernant. L'obtention de ces toxines par voie chimique ou recombinante, l'introduction de résidus non naturels au sein de leurs séquences et la connaissance structurale de leur site d'interaction permet d'envisager la conception de nouveaux ligands ayant des sélectivités prédéfinies et pouvant avoir un intérêt en tant qu'outils pharmacologiques et/ou agents thérapeutiques dans les nombreuses pathologies impliquant ces récepteurs

How do short neurotoxins bind to a muscular-type nicotinic acetylcholine receptor?

International audienceWe investigated the interacting surface between a short curarimimetic toxin and a muscular-type nicotinic acetylcholine receptor, looking for the ability of various biotinylated Naja nigricollis ␣-neurotoxin analogues to bind simultaneously the receptor and streptavidin. All these derivatives, modified at positions 10 (loop I), 27, 30, 33, 35 (loop II), 46, and 47 (loop III) or the N-terminal (erabutoxin numbering), still shared high affinity for the receptor, and in the absence of receptor they all bound soluble streptavidin. However, the proportion of the toxin-receptor complex that bound to streptavidincoated beads, varied both with the location of the modification and with the length of the linker between biotin and the toxin. In the receptor-toxin complex, the concave side of loops II and III was not accessible to streptavidin, unlike the N terminus of the toxin and, to a certain extent, loop I. On the convex face, loop III was the most accessible, whereas the tip of loop II, especially Arg-30, seemed to be closer to the receptor. The present data demonstrate that short toxins neither penetrate deeply into a crevice as proposed earlier nor lie parallel to the receptor extracellular wall. These data also suggest that they may not lie strictly perpendicular to the cylindrical wall of the receptor. These results fit nicely with three-dimensional models of interaction between long neurotoxins and their receptors and support the idea that short and long curarimimetic toxins share a similar overall topology of interaction when bound to nicotinic receptors

A Cysteine-Linkable, Short Cleavable Photoprobe with Dual Functionality To Explore Protein−Protein Interfaces

International audienc

Chemical engineering of a three-fingered toxin with anti-α7 neuronal acetylcholine receptor activity

International audienc

Motions and structural variability within toxins: Implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the “three-finger” fold and the short α/β scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short α/β scorpion fold is highly compact, whereas the “three-finger” fold is a β structure presenting large flexible loops. First, the crystal structure of the snake toxin α was solved at 1.8-Å resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin α and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin α. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions