Topography of toxin-acetylcholine receptor complexes by using photoactivatable toxin derivatives.

Abstract

We have defined the molecular environment of a snake neurotoxin interacting with the high-and lowaffinity binding sites of the nicotinic acetylcholine receptor (AcChoR). This was done by photocoupling reactions using three toxin derivatives with photoactivatable moieties on Lys-15, Lys-47, and Lys-51. Competition data showed that Lys47 belongs to the toxin-AcChoR interacting domain whereas the other two residues are excluded from it. We first tentatively determined the threshold of covalent coupling, indicative of the proximity between the photoactivatable probes and subunits, by quantifying the coupling occurring between the same derivatives and a model compound (i.e., a toxin-specific monoclonal antibody). We then (i) quantified the coupling yields occurring when both binding sites of AcChoR were occupied by the toxin derivatives, (ii) discriminately quantified the coupling yields at the high-affinity binding site, and (iii) deduced the coupling yields at the low-affinity binding site. In the highaffinity site, the probes on Lys-15 and Lys47 predominantly reacted with the high-affinity site of the AcChoR a subunit whereas the probe on Lys-51 reacted with the 8 subunit. In the low-affinity site, the probe on Lys-47 predominantly reacted with the low-affinity site of the a chain and the I8 chain whereas those on Lys-15 and Lys-51 reacted with the y and 8 chains, respectively. A three-dimensional model showing a unique organization of AcChoR bound to two toxin molecules is presented.Determination of protein-protein interactions is one of the most challenging issues in biology. Interacting domains can be identified by chemical modifications of selected residues. Such techniques have proven suitable for delineating functionally critical residues of curaremimetic toxins from snake venoms (1-11). The domain by which toxin-a, a neurotoxin present in venom of the spitting cobra Naja nigricollis (12) MATERIALS AND METHODS HPLC columns C18 p.Bondapak were purchased from Waters. Toxin-a from N. nigricollis was prepared from venom as described (12). Torpedo marmorata AcChoR-rich membranes were purified according to Saitoh et al. (15). The monoclonal antibody (Mal) was prepared and purified according to Boulain et al. (8). The concentration of toxin-a binding sites was determined using [3H]toxin-a as a radioactive tracer. All other chemicals were of the purest grade commercially available.The preparation and characterization of photoactivatable derivatives will be published in detail elsewhere. Briefly, 1 tmol of [3H]toxin-a (3 Ci/mmol; 1 Ci = 37 GBq) in 0.5 ml of 0.05 M sodium phosphate (pH 7) was incubated with 3 Amol of p-azidobenzoyl N-hydroxysuccinimide ester in 0.5 ml of acetonitrile for 3 hr at room temperature (10). The toxin derivatives were separated from excess reagent by filtration through Bio-Gel P-2 (1 cm x 18 cm) equilibrated in 10%6 (vol/vol) acetic acid and fractionated on a C18 reversed-phase HPLC column. Four major radioactive fractions were resolved. Their molar extinction coefficient determined ...

Allosteric transitions of the acetylcholine receptor probed at the amino acid level with a photolabile cholinergic ligand.

“…The amino acids labeled by DDF belong to three distinct peptide segments of the large amino-terminal hydrophilic domain of the a subunit, which include Tyr-93, Trp-149, 15), and the two cysteines, 192 and 193, initially identified (16) with a maleimido derivative. Furthermore, DDF also reacts, though to a smaller extent, with the ,B, y, and 8 subunits (13), suggesting that they may each participate in one of the two binding areas in conjunction with one a subunit (17)(18)(19)(20) and thereby account for the distinct binding specificity of the two a subunits (21,22).…”

Yeast Expression and NMR Analysis of the Extracellular Domain of Muscle Nicotinic Acetylcholine Receptor α Subunit

“…The neurotransmitter ACh interacts with two nonequivalent sites near the interface of the ␣␦ and ␣␥ subunits (2,11,12). The binding site for ␣-bungarotoxin (␣-BuTx) is located largely on the amino-terminal extracellular domain of the ␣ subunit (13)(14)(15)(16). In addition, this part of the subunit also possesses the main immunogenic region, which stimulates the production and forms the binding site for autoimmune antibodies in sera of patients with myasthenia gravis (17).…”

Interaction of modified neurotoxins fromNaja nigricollis with the nicotinic acetylcholine receptor fromTorpedo marmorata A Raman spectroscopy study

“…An important issue in the understanding at the molecular level of the mode of action of the neurotoxins is the identification of the side-chains involved in the binding sites of both molecules. Studies of the interaction of the toxin c~ from N. nigricollis using EPR [9] tolabelling experiments [12] have been reported. Amino acids which belong to the binding site for cholinergic ligands on the cz-subunit have been identified by means of affinity reagents which covalently bind to the acetylcholine binding site [13,14] or by binding of the 0t-neurotoxin to proteolytic fragments of the ~-subunit [15][16][17].…”