Waglerin-1 (Wtx-1) is a 22-amino acid peptide that is a competitive antagonist of the muscle nicotinic receptor (nAChR). We find that Wtx-1 binds 2100-fold more tightly to the α-ε than to the α-δ binding site interface of the mouse nAChR. Moreover, Wtx-1 binds 100-fold more tightly to the α-ε interface from mouse nAChR than that from rat or human sources. Site-directed mutagenesis of residues differing in the extracellular domains of rat and mouse ε subunits indicates that residues 59 and 115 mediate the species difference in Wtx-1 affinity. Mutation of residues 59 (Asp in mouse, Glu in rat ε) and 115 (Tyr in mouse, Ser in rat ε) converts Wtx-1 affinity for the α-ε interface of one species to that of the other species. Studies of different mutations at position 59 indicate both steric and electrostatic contributions to Wtx-1 affinity, whereas at position 115, both aromatic and polar groups contribute to affinity. The human nAChR also has lower affinity for Wtx-1 than mouse nAChR, but unlike rat nAChR, residues in both α and ε subunits mediate the affinity difference. In human nAChR, polar residues (Ser-187 and Thr-189) confer low affinity, whereas in mouse nAChR aromatic residues (Trp-187 and Phe-189) confer high affinity. The overall results show that non-conserved residues at the nAChR binding site, although not crucial for activation by ACh, govern the potency of neuromuscular toxins.
The three-fingered α-neurotoxins isolated from the venoms of Elapidae family snakes have long been used as tools for the isolation and characterization of the nicotinic acetylcholine receptor (nAChR). These small proteins cause paralysis and death by binding to the nAChR at the neuromuscular junction and competitively antagonizing the action of acetylcholine. Snakes of the Viperidae family were not previously known to make toxins that target nAChR. A decade ago, four peptides of 22–24 amino acids were characterized from the venom of Wagler's Pit Viper, Tropidolaemus wagleri. These peptides are unique among the lethal components of Viperid venoms in that their lethal effects, like the three-fingered toxins, are mediated by competitive antagonism of muscle nAChR. One of these peptides, called Waglerin-1, also possesses a distinctive selectivity for the α–ε interface binding site of the mouse nAChR, binding with over 2000-fold higher affinity to the α–ε site compared to the α–δ or α–γ binding sites. Furthermore, it binds the α–ε interface site of cloned mouse nAChR with 100-fold higher affinity than both the rat or the human cloned receptors. The amino acid residues of the receptor mediating both the binding site and species selectivity have been determined. Additional work on the structure of the peptide itself has begun to elucidate the residues of the peptide most responsible for its high affinity and selectivity. The peptides produced by this unique snake provide researchers with a new structural template for studying nAChR and other receptors. Additional research into the low molecular weight venom components of T. wagleri and its cousins in the Viperidae family should yield new structural information on receptors and toxins.
alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.
αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.
