Engineering of Three-Finger Fold Toxins Creates Ligands with Original Pharmacological Profiles for Muscarinic and Adrenergic Receptors

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test “loop grafting,” a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α1A-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α1A-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles

Identification and Structural Characterization of a New Three-Finger Toxin Hemachatoxin from Hemachatus haemachatus Venom

10.1371/journal.pone.0048112PLoS ONE710

Quels sont les déterminants moléculaires impliqués dans la spécificité d'interaction de neurotoxines pour les récepteurs nicotiniques de type musculaire et neuronal de type alpha7 ?

PARIS-BIUP (751062107) / SudocSudocFranceF

Molecular Determinants by Which a Long Chain Toxin from

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Molecular Determinants by Which a Long Chain Toxin from Snake Venom Interacts with the Neuronal α7-Nicotinic Acetylcholine Receptor

International audienc

Molecular Characterization of the Specificity of Interactions of Various Neurotoxins on Two Distinct Nicotinic Acetylcholine Receptors

Snake curaremimetic toxins are currently classified as short-chain and long-chain toxins according to their size and their number of disulfide bonds. All these toxins bind with high affinity to muscular-type nicotinic acetylcholine receptor, whereas only long toxins recognize the a7 receptor with high affinity. On the basis of binding experiments with Torpedo or neuronal a7 receptors using wild-type and mutated neurotoxins, we characterized the molecular determinants involved in these different recognition processes. The functional sites by which long and short toxins interact with the muscular-type receptor include a common core of highly conserved residues and residues that are specific to each of toxin families. Furthermore, the functional sites through which a-cobratoxin, a long-chain toxin, interacts with muscular and a7 receptors share similarities but also marked differences. Our results reveal that the three-finger fold toxins have evolved toward various specificities by displa..

Experimentally based model of a complex between a snake toxin and the 7 nicotinic receptor

International audienceTo understand how snake neurotoxins interact with nicotinic acetylcholine receptors, we have elaborated an experimentally based model of the alpha-cobratoxin-alpha7 receptor complex. This model was achieved by using (i) a three-dimensional model of the alpha7 extracellular domain derived from the crystallographic structure of the homologous acetylcholine-binding protein, (ii) the previously solved x-ray structure of the toxin, and (iii) nine pairs of residues identified by cycle-mutant experiments to make contacts between the alpha-cobratoxin and alpha7 receptor. Because the receptor loop F occludes entrance of the toxin binding pocket, we submitted this loop to a dynamics simulation and selected a conformation that allowed the toxin to reach its binding site. The three-dimensional structure of the toxin-receptor complex model was validated a posteriori by an additional double-mutant experiment. The model shows that the toxin interacts perpendicularly to the receptor axis, in an equatorial position of the extracellular domain. The tip of the toxin central loop plugs into the receptor between two subunits, just below the functional receptor loop C, the C-terminal tail of the toxin making adjacent additional interactions at the receptor surface. The receptor establishes major contacts with the toxin by its loop C, which is assisted by principal (loops A and B) and complementary (loops D, F, and 1) functional regions. This model explains the antagonistic properties of the toxin toward the neuronal receptor and opens the way to the design of new antagonists

Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain α-neurotoxins have a CTX-like cytolytic activity