The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors

Abstract

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

A current perspective on snake venom composition and constituent protein families

“…These non-enzymatic toxins have a mass of between 6 and 9 kDa (Clarke et al 2006), and are named after their characteristic spatial structure, as all have a similar highly conserved folding pattern consisting of three β-stranded loops (fingers), extending from a globular central core which is stabilised by four conserved disulfide bridges (Kini and Doley 2010). An important sub-group of this protein family are the α-neurotoxins, post-synaptic neurotoxins that act at the nicotinic acetylcholine receptors (nAChRs) (Nys et al 2022). Alpha-neurotoxins competitively antagonise the ligand-gated ion channels at the acetylcholine binding site and prevent the opening of the channel.…”

Membrane-mediated interaction of non-conventional snake three-finger toxins with nicotinic acetylcholine receptors

“…Interaction of the short-chain neurotoxin NTII from Naja oxiana (UniProt ID: P01427), which has three positively charged residues in the corresponding region, with model lipid membranes was demonstrated previously 44 . Despite this, the recent cryo-EM structure of the prototypical short-chain neurotoxin ScNtx, a homolog of NTII, in complex with muscle-type nAChR 27 , like the earlier complexes of long-chain α-Bgtx 16 , 26 , did not reveal contacts with the membrane environment of the receptor. Thus, the presence of a membrane-binding site in the toxin molecule and interaction with the membrane in the absence of a receptor does not obligatorily indicate the presence of contacts with the lipid bilayer in the toxin-nAChR complex.…”

“…Similar modes of the α-Bgtx interaction were observed in the complexes with full-length α7-nAChR 16 and muscle type nAChR from Torpedo californica 26 . Interestingly, the recent structure of the complex of the prototypical synthetic short-chain α-neurotoxin ScNtx with Torpedo nAChR 27 also demonstrates the similar interaction topology, where loop II of the toxin penetrates under the receptor’s loop C into the orthosteric site, and the loop C of the receptor gets caught between the loops I and II of the toxin. Probably, this interaction mode is common for the complexes of long-chain and short-chain α-neurotoxins with various subtypes of nAChR.…”

“…A typical representative of the latter group is the weak toxin from Naja kaouthia venom (WTX), which demonstrates a low affinity both for muscle-type and α7-type nAChRs in the range of tens of µM 23 – 25 . To date, the structures of short-chain and long-chain neurotoxins in complex with muscle-type or α7-type nAChRs were determined by cryo-EM 16 , 26 , 27 . In contrast, no structures of the complexes of non-conventional toxins with nAChRs are known to date.…”

Domain loss enabled evolution of novel functions in a gene superfamily

“…1E) exhibit increased potency, which likely contributed to explosive diversification of elapids [9]. The structural integrity conferred by simplification of these toxins facilitated an accumulation of mutations in their surface residues, with consequent diversification of their interaction partners [13]. Various short-chain 3FTxs bind and modulate a range of targets, including muscarinic acetylcholine receptors, adrenergic receptors, L-type calcium channels, and voltage-gated sodium channels.…”