Structural Insight into Specificity of Interactions between Nonconventional Three-finger Weak Toxin from Naja kaouthia (WTX) and Muscarinic Acetylcholine Receptors

Ekaterina N Lyukmanova,Zakhar O Shenkarev,Mikhail A Shulepko,Alexander S Paramonov,Anton O Chugunov,Helena Janickova,Eva Dolejsi,Vladimir Dolezal,Yuri N Utkin,Victor I Tsetlin,Alexander S Arseniev,Roman G Efremov,Dmitry A Dolgikh,Mikhail P Kirpichnikov

doi:10.1074/jbc.m115.656595

Abstract

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.

Highlights

Cobra’s “three-finger” nonconventional toxin Weak toxin from Naja kaouthia (WTX) allosterically modulates muscarinic receptors
WTX irreversibly binds to muscle and ␣7 nicotinic acetylcholine receptors (nAChRs) with IC50 values in the micromolar range [14] and could allosterically interact with different subtypes of muscarinic acetylcholine receptors (mAChRs) modulating binding of orthosteric ligands, like acetylcholine and N-methylscopolamine (NMS) [15]
Computer Modeling of recombinant mutant WTX analogue (rWTX)(P33A) Interaction with M1 and M3 mAChRs—In an attempt to understand the mechanisms of target-specific positive and negative allosteric rWTX activity, we modeled the complexes of the rWTX(P33A) mutant with M1- and M3-mAChRs

Summary

Background

Cobra’s “three-finger” nonconventional toxin WTX allosterically modulates muscarinic receptors (mAChRs). 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). Separate groups of three-finger toxins act on numerous targets [2], including two classes of acetylcholine receptors as follows: nicotinic acetylcholine receptors (nAChRs) belonging to ligand-gated ion channels [3] and muscarinic acetylcholine receptors (mAChRs) of the G protein-coupled receptor (GPCR) family [4]. Nonconventional “weak” toxin from Naja kaouthia (WTX) is nonlethal to mice at concentrations up to 20 mg/kg and shares some biological properties with ␣-NTs and MTs. WTX irreversibly binds to muscle and ␣7 nAChRs with IC50 values in the micromolar range [14] and could allosterically interact with different subtypes of mAChRs modulating binding of orthosteric ligands, like acetylcholine and N-methylscopolamine (NMS) [15]. A significant conformational plasticity of the loop II observed by NMR and molecular dynamics (MD) simulations could explain the WTX activity toward structurally unrelated targets, nAChRs and mAChRs

Experimental Procedures

Results

Discussion