Abstract
Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M1 muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M1 receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M5 subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M1 receptor. An M5 receptor construct (M5-E175Y184E474), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent Ki = 24 nM; apparent Ki for M1 = 0.5 nM). After screening for different domains and residues, we found a specific residue (P179 to L in M5) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M5-EL179YE, apparent Ki = 0.5 nM). Mutation of P179 to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.
Highlights
Muscarinic acetylcholine receptors are G protein-coupled receptors that mediate the metabotropic effects of acetylcholine in vertebrates as well as in invertebrates
CH2) indicated that we should refocus on the ECL2, since CH2 incorporating the ECL2 of M1 increased the affinity for Muscarinic toxin 7 (MT7) about tenfold as compared to M5-EYE (Table 1)
After screening for individual residues in ECL2 of M5, we found that the additional P179 to L (M5-ELYE) mutation increased significantly the affinity for MT7 (Figure 2a)
Summary
Muscarinic acetylcholine receptors (mAChRs) are G protein-coupled receptors that mediate the metabotropic effects of acetylcholine in vertebrates as well as in invertebrates. MT7, called m1-toxin, is unique in the respect that it shows full selectivity for the M1 subtype, i.e., subnanomolar affinity for M1 and no detectable binding to the other subtypes at micromolar concentrations [15,16] 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].
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