Weak toxin from Naja kaouthia (WTX) belongs to the group of nonconventional "three-finger" snake neurotoxins. It irreversibly inhibits nicotinic acetylcholine receptors and allosterically interacts with muscarinic acetylcholine receptors (mAChRs). Using site-directed mutagenesis, NMR spectroscopy, and computer modeling, we investigated the recombinant mutant WTX analogue (rWTX) which, compared with the native toxin, has an additional N-terminal methionine residue. In comparison with the wild-type toxin, rWTX demonstrated an altered pharmacological profile, decreased binding of orthosteric antagonist N-methylscopolamine to human M1- and M2-mAChRs, and increased antagonist binding to M3-mAChR. Positively charged arginine residues located in the flexible loop II were found to be crucial for rWTX interactions with all types of mAChR. Computer modeling suggested that the rWTX loop II protrudes to the M1-mAChR allosteric ligand-binding site blocking the entrance to the orthosteric site. In contrast, toxin interacts with M3-mAChR by loop II without penetration into the allosteric site. Data obtained provide new structural insight into the target-specific allosteric regulation of mAChRs by "three-finger" snake neurotoxins.

From the Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences 16 10 Miklukho Maklaya Street 117997 Moscow Russia

From the Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences 16 10 Miklukho Maklaya Street 117997 Moscow Russia the Lomonosov Moscow State University 119991 Moscow Russia

From the Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences 16 10 Miklukho Maklaya Street 117997 Moscow Russia the Lomonosov Moscow State University 119991 Moscow Russia the Moscow Institute of Physics and Technology Institutskiy Pereulok 9 Dolgoprudny Moscow Region 141700 Russian

From the Shemyakin Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences 16 10 Miklukho Maklaya Street 117997 Moscow Russia the Moscow Institute of Physics and Technology Institutskiy Pereulok 9 Dolgoprudny Moscow Region 141700 Russian

the Institute of Physiology Academy of Sciences of the Czech Republic 14220 Prague Czech Republic and

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PDB
2MJ0, 4DAJ