Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein
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 mutage...
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doaj-39ad45eaf5854319bc2c438bb6a0748e2020-11-24T23:47:48ZengMDPI AGToxins2072-66512011-11-013111393140410.3390/toxins3111393Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding ProteinKatja NäreojaJohnny NäsmanSergio RondinelliMuscarinic 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.http://www.mdpi.com/2072-6651/3/11/1393/G protein-coupled receptormuscarinic toxinacetylcholine receptorligand binding |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Katja Näreoja Johnny Näsman Sergio Rondinelli |
spellingShingle |
Katja Näreoja Johnny Näsman Sergio Rondinelli Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein Toxins G protein-coupled receptor muscarinic toxin acetylcholine receptor ligand binding |
author_facet |
Katja Näreoja Johnny Näsman Sergio Rondinelli |
author_sort |
Katja Näreoja |
title |
Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein |
title_short |
Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein |
title_full |
Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein |
title_fullStr |
Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein |
title_full_unstemmed |
Molecular Conversion of Muscarinic Acetylcholine Receptor M5 to Muscarinic Toxin 7 (MT7)-Binding Protein |
title_sort |
molecular conversion of muscarinic acetylcholine receptor m5 to muscarinic toxin 7 (mt7)-binding protein |
publisher |
MDPI AG |
series |
Toxins |
issn |
2072-6651 |
publishDate |
2011-11-01 |
description |
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. |
topic |
G protein-coupled receptor muscarinic toxin acetylcholine receptor ligand binding |
url |
http://www.mdpi.com/2072-6651/3/11/1393/ |
work_keys_str_mv |
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