Snake α-Neurotoxin Binding Site on the Egyptian Cobra (Naja haje) Nicotinic Acetylcholine Receptor Is Conserved

Abstract

Evolutionary success requires that animal venoms are targeted against phylogenetically conserved molecular structures of fundamental physiological processes. Species producing venoms must be resistant to their action. Venoms of Elapidae snakes (e.g., cobras, kraits) contain alpha-neurotoxins, represented by alpha-bungarotoxin (alpha-BTX) targeted against the nicotinic acetylcholine receptor (nAChR) of the neuromuscular junction. The model which presumes that cobras (Naja spp., Elapidae) have lost their binding site for conspecific alpha-neurotoxins because of the unique amino acid substitutions in their nAChR polypeptide backbone per se is incompatible with the evolutionary theory that (1) the molecular motifs forming the alpha-neurotoxin target site on the nAChR are fundamental for receptor structure and/or function, and (2) the alpha-neurotoxin target site is conserved among Chordata lineages. To test the hypothesis that the alpha-neurotoxin binding site is conserved in Elapidae snakes and to identify the mechanism of resistance against conspecific alpha-neurotoxins, we cloned the ligand binding domain of the Egyptian cobra (Naja haje) nAChR alpha subunit. When expressed as part of a functional Naja/mouse chimeric nAChR in Xenopus oocytes, this domain confers resistance against alpha-BTX but does not alter responses induced by the natural ligand acetylcholine. Further mutational analysis of the Naja/mouse nAChR demonstrated that an N-glycosylation signal in the ligand binding domain that is unique to N. haje is responsible for alpha-BTX resistance. However, when the N-glycosylation signal is eliminated, the nAChR containing the N. haje sequence is inhibited by alpha-BTX with a potency that is comparable to that in mammals. We conclude that the binding site for conspecific alpha-neurotoxin in Elapidae snakes is conserved in the nAChR ligand binding domain polypeptide backbone per se. This conclusion supports the hypothesis that animal toxins are targeted against evolutionarily conserved molecular motifs. Such conservation also calls for a revision of the present model of the alpha-BTX binding site. The approach described here can be used to identify the mechanism of resistance against conspecific venoms in other species and to characterize toxin-receptor coevolution.

Structural Determinants for α-Neurotoxin Sensitivity in Muscle nAChR and Their Implications for the Gating Mechanism

“…7,9 Furthermore, two studies showed that the α1 subunit of the muscle-type nAChR bears the major structural determinants for α-neurotoxins sensitivity. 10,11 This is consistent with the fact that most of the structural elements constituting the antagonist-binding site in nAChRs are located in the α subunits. 3,4 Ion channels are major pharmaceutical targets, 12 and nAChRs are of particular relevance to a variety of diseases including Parkinson's, Alzheimer's, myasthenia gravis, schizophrenia, epilepsy, depression and substance addiction.…”

“…As revealed by our crystal structure, 4 most of the sequence variations implicated in modulating the receptor's sensitivity to α-neurotoxins are indeed located at or near the receptor/ toxin interface [7][8][9][10][11]14,15 ( Fig. 2A and B).…”

“…10,11 However, as discussed below, most of these changes, except for Tyr112His in mongoose, are located at positions that affect α-Btx binding either directly or indirectly (Fig. 2B).…”

“…Gly153Ser may also alter the main chain conformation of loop B and affect the receptor's binding affinity for acetylcholine. 16 In snakes, Asn189 constitutes a functional glycosylation site that introduces a bulky glycan right at the center of the toxin/receptor interface, 7,8,11 which seems to be the most effective way to block toxin binding through steric clashes.…”

“…However, since residue 187 is located at the periphery of the toxin/receptor interface, a glycan chain introduced at this site may not be as effective in blocking toxin binding as that found in snakes at position 189. 11,14,15 It is important to note that while the conserved glycan on the Cys-loop contributes positively to toxin binding, the glycans introduced at Asn189 in snakes and Asn187 in mongoose likely interfere with toxin binding due to steric clashes.…”