Different Interactions between MT7 Toxin and the Human Muscarinic M₁ Receptor in Its Free and N-Methylscopolamine-Occupied States

Abstract

Muscarinic MT7 toxin is a highly selective and potent antagonist of the M 1 subtype of muscarinic receptor and acts by binding to an allosteric site. To identify the molecular determinants by which MT7 toxin interacts with this receptor in its free and NMS-occupied states, the effect on toxin potency of alanine substitution was evaluated in equilibrium and kinetic binding experiments as well as in functional assays. The determination of the crystallographic structure of an MT7-derivative (MT7-diiodoTyr51) allowed the selection of candidate residues that are accessible and present on both faces of the three toxin loops. The equilibrium binding data are consistent with negative cooperativity between N-methylscopolamine (NMS) and wild-type or modified MT7 and highlight the critical role of the tip of the central loop of the toxin (Arg34, Met35Tyr36) in its interaction with the unoccupied receptor. Examination of the potency of wild-type and modified toxins to allosterically decrease the dissociation rate of [ 3 H]NMS allowed the identification of the MT7 residues involved in its interaction with the NMSoccupied receptor. In contrast to the results with the unoccupied receptor, the most important residue for this interaction was Tyr36 in loop II, assisted by Trp10 in loop I and Arg52 in loop III. The critical role of the tips of the MT7 loops was also confirmed in functional experiments. The high specificity of the MT7-M 1 receptor interaction exploits several MT7-specific residues and reveals a different mode of interaction of the toxin with the free and NMS-occupied states of the receptor.Muscarinic neurotoxins, small peptides of 64 to 66 residues derived from the venom of African mambas (Dendroaspis angusticeps and Dendroaspis polylepis), are well known for their ability to interact with different muscarinic receptor subtypes. NMR and X-ray studies of the MT2 toxin have shown that muscarinic toxins have the three-finger fold structure, characteristic of the large superfamily of toxins that act at cholinergic synapses (Ménez and Ducruix, 1993;Ségalas et al., 1995;Servent and Ménez, 2001). Ten different muscarinic toxins have been isolated so far. Despite their high sequence homology, they show divergent interaction profiles with the different muscarinic receptor subtypes (for review, see Bradley, 2000;Karlsson et al., 2000). For instance, the MT7 toxin, purified from the venom of the green mamba, binds to the M 1 receptor subtype with a potency in the low or subnanomolar range, with at least 10,000-fold selectivity relative to the other receptor subtypes (Max et al., 1993;Carsi and Potter, 2000;Olianas et al., 2000;Mourier et al., 2003 (Olianas et al., 2000(Olianas et al., , 2004Krajewski et al., 2001;Mourier et al., 2003). In summary, MT7 acts as a highly selective antagonist of M 1 receptors by establishing a strong and stable interaction with an allosteric binding site on the receptor.There is a limited understanding of the specificity, selectivity, and mechanism of action of the muscarinic toxins at Ar...

Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein

“…Since this toxin can discriminate so well among mAChR subtypes, it would be of general interest for ligand-receptor interactions to uncover the binding mechanism for MT7. It has been shown by site-directed mutagenesis of MT7 that many amino acid residues seem to make up the total binding affinity, and these residues are distributed on all three finger loops of the toxin [17]. Regarding the corresponding sites on the receptor, it can be anticipated that binding contacts with loosely conserved regions are crucially involved in the selectivity.…”

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins

“…4A). Superimposition on other known mamba toxins, MT1 (PDB id: 4DO8 23 ), MT2 (PDB id: 1FF4), MT7 (PDB id: 2VLW 33 ) and ρ-Da1a (PDB id: 4IYE 34 ) showed strong conservation of the backbone (Fig. 4B).…”

“…The crystallization trials were carried out using Cryst Chem TM sitting drop vapor diffusion plates with 1 µl drops of protein and precipitant, stored in a constant temperature incubator at 20 °C. Limited screening for crystals was carried out starting from conditions used for MT7 33 . Crystals of AncTx1-W28R/I38S were obtained from 1.9 M ammonium sulfate, 4% MPD, 2% 1,4-dioxane, 2% gamma-valerolactone, 0.132 M sodium citrate, pH 5.5.…”

All muscarinic acetylcholine receptors (M1-M5) are expressed in murine brain microvascular endothelium

“…The functional expression of mAChRs in BMVECs and bEnd.3 cells was also evaluated by applying agonists/antagonists targeting the allosteric site of the receptors. We found that the selective allosteric M 1 antagonist MT-7 (20 nM) 38 did not exert any inhibitory effect on the ACh-induced calcium signal in either type of brain microvascular endothelial cells (Supplementary Fig. 7A).…”