Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis.

Pillet, Laurence; Trémeau, O.; Ducancel, Frédéric; Drevet, Pascal; Zinn‐Justin, Sophie; Pinkasfeld, Suzanne; Boulain, Jean‐Claude; Ménèz, Andre

doi:10.1016/s0021-9258(18)54020-5

Cited by 150 publications

(59 citation statements)

References 59 publications

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“…These 'pharmacophore' residues that interact with Torpedo (α1) 2 βγδ nAChRs are identical in both toxins, and are located at homologous positions in loop II. The 'muscle' pharmacophore comprises, respectively, [10,[47][48][49][50].…”

Section: Discussionmentioning

confidence: 99%

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)2βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

Blacklow

Kornhauser

Hains

et al. 2011

Biochemical Pharmacology

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In contrast to all classical long-chain α-neurotoxins possessing the critical fifth disulfide bond, α-elapitoxin-Aa2a (α-EPTX-Aa2a), a novel long-chain α-neurotoxin from the common death adder Acanthophis antarcticus, lacks affinity for neuronal α7-type nicotinic acetylcholine receptors (nAChRs). α-EPTX-Aa2a (8850Da; 0.1-1μM) caused a concentration-dependent inhibition of indirect twitches, and blocked contractures to cholinergic agonists in the isolated chick biventer cervicis nerve-muscle preparation, consistent with a postsynaptic curaremimetic mode of action. α-EPTX-Aa2a (1-10nM) produced a potent pseudo-irreversible antagonism of chick muscle nAChRs, with an estimated pA(2) value of 8.311±0.031, which was not reversed by monovalent death adder antivenom. This is only 2.5-fold less potent than the prototypical long-chain α-neurotoxin, α-bungarotoxin. In contrast, α-EPTX-Aa2a produced complete, but weak, inhibition of (125)I-α-bungarotoxin binding to rat hippocampal α7 nAChRs (pK(I)=3.670), despite high sequence homology and similar mass to a wide range of long-chain α-neurotoxins. The mostly likely cause for the loss of α7 binding affinity is a leucine substitution, in loop II of α-EPTX-Aa2a, for the highly conserved Arg(33) in long-chain α-neurotoxins. Arg(33) has been shown to be critical for both neuronal and muscle activity. Despite this substitution, α-EPTX-Aa2a retains high affinity for muscle (α1)(2)βγδ nAChRs. This is probably as a result of an Arg(29) residue, previously shown to be critical for muscle (α1)(2)βγδ nAChR affinity, and highly conserved across all short-chain, but not long-chain, α-neurotoxins. α-EPTX-Aa2a therefore represents a novel atypical long-chain α-neurotoxin that includes a fifth disulfide but exhibits differential affinity for nAChR subtypes.

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Section: Discussionmentioning

confidence: 99%

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)2βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

Blacklow

Kornhauser

Hains

et al. 2011

Biochemical Pharmacology

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“…It was found that the toxin interacted with the binding sites of α7 and muscle-type nAChR through D31, F32, and R33 amino acid residues. All these interactions were shown earlier to be important for erabutoxin a binding to the muscle-type nAChR [ 213 ].…”

Section: Marine Protein Ligands Of Nachr—still An Open Field For Rese...mentioning

confidence: 99%

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

et al. 2022

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The purpose of our review is to briefly show what different compounds of marine origin, from low molecular weight ones to peptides and proteins, offer for understanding the structure and mechanism of action of nicotinic acetylcholine receptors (nAChRs) and for finding novel drugs to combat the diseases where nAChRs may be involved. The importance of the mentioned classes of ligands has changed with time; a protein from the marine snake venom was the first excellent tool to characterize the muscle-type nAChRs from the electric ray, while at present, muscle and α7 receptors are labeled with the radioactive or fluorescent derivatives prepared from α-bungarotoxin isolated from the many-banded krait. The most sophisticated instruments to distinguish muscle from neuronal nAChRs, and especially distinct subtypes within the latter, are α-conotoxins. Such information is crucial for fundamental studies on the nAChR revealing the properties of their orthosteric and allosteric binding sites and mechanisms of the channel opening and closure. Similar data are provided by low-molecular weight compounds of marine origin, but here the main purpose is drug design. In our review we tried to show what has been obtained in the last decade when the listed classes of compounds were used in the nAChR research, applying computer modeling, synthetic analogues and receptor mutants, X-ray and electron-microscopy analyses of complexes with the nAChRs, and their models which are acetylcholine-binding proteins and heterologously-expressed ligand-binding domains.

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“…An alternative for venom purified α-3FNTxs was found in their heterologous expression in bacteria or yeast [39,40], which facilitated seminal mutagenesis work by Menez and co-workers to decipher the pharmacophores of α-3FNTxs [41,42]; as well as with chemical synthesis of complex 3FTxs [43][44][45]. The utilisation of "mimotope" α-3FNTx fragments that bind nAChRs [46][47][48], including combinatorial high-affinity peptides (HAPs) [49] have provided valuable insight into toxinreceptor interactions, as well as allowing their use as epitopes to tag and study other membrane receptors such as AMPA and GABAB [50,51].…”

Section: Historical Perspectives Of Three-finger α-Neurotoxin Research Milestonesmentioning

confidence: 99%

“…Other residues, Gly42, Pro44 and Pro48 were also important to maintain the 3FP conformation, whereas Gly40 enabled close packing of the protein [22,123,140]. Likewise, the conserved Ser8 in EbTx-a is believed to be crucial in stabilising the orientation of side chains in Loop II, thus indirectly contributing to its function [41]. Some charged residues, Arg39 in EbTxa and Asp60 in α-CbTx for instance, were shown to stabilize the native conformation of the 3FP by forming salt links with the C-or N-terminus of the toxin [123,195].…”

Section: Structural Stability Of the Three-finger Protein Foldmentioning

confidence: 99%

Scope of practice and educational needs of infection prevention and control professionals in Australian residential aged care facilities

Shaban

Sotomayor-Castillo

Macbeth

et al. 2020

Infection, Disease & Health

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Snake venom three-finger α-neurotoxins (α-3FNTx) act on postsynaptic nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction (NMJ) to produce skeletal muscle paralysis.The discovery of the archetypal α-bungarotoxin (α-BgTx), almost six decades ago, exponentially expanded our knowledge of membrane receptors and ion channels. This included the localisation, isolation and characterization of the first receptor (nAChR); and by extension, the pathophysiology and pharmacology of neuromuscular transmission and associated pathologies such as myasthenia gravis, as well as our understanding of the role of α-3FNTxs in snakebite envenomation leading to novel concepts of targeted treatment. Subsequent studies on a variety of animal venoms have yielded a plethora of novel toxins that have revolutionized molecular biomedicine and advanced drug discovery from bench to bedside. This review provides an overview of nAChRs and their subtypes, classification of α-3FNTxs and the challenges of typifying an increasing arsenal of structurally and functionally unique toxins, and the three-finger protein (3FP) fold in the context of the uPAR/Ly6/CD59/snake toxin superfamily. The pharmacology of snake α-3FNTxs including their mechanisms of neuromuscular blockade, variations in reversibility of nAChR interactions, specificity for nAChR subtypes or for distinct ligand-binding interfaces within a subtype and the role of α-3FNTxs in neurotoxic envenomation are also detailed. Lastly, a reconciliation of structure-function relationships between α-3FNTx and nAChRs, derived from historical mutational and biochemical studies and emerging atomic level structures of nAChR models in complex with α-3FNTxs is discussed.

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Genetic engineering of snake toxins. Role of invariant residues in the structural and functional properties of a curaremimetic toxin, as probed by site-directed mutagenesis.

Cited by 150 publications

References 59 publications

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)2βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

α-Elapitoxin-Aa2a, a long-chain snake α-neurotoxin with potent actions on muscle (α1)2βγδ nicotinic receptors, lacks the classical high affinity for neuronal α7 nicotinic receptors

Marine Origin Ligands of Nicotinic Receptors: Low Molecular Compounds, Peptides and Proteins for Fundamental Research and Practical Applications

Scope of practice and educational needs of infection prevention and control professionals in Australian residential aged care facilities

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