title: A Distinct Functional Site in Ω-Neurotoxins: Novel Antagonists of Nicotinic Acetylcholine Receptors from Snake Venom.
abstract: Snake venom α-neurotoxins from the three-finger toxin (3FTx) family are competitive antagonists with nanomolar affinity and high selectivity for nicotinic acetylcholine receptors (nAChR). Here, we report the characterization of a new group of competitive nAChR antagonists: Ω-neurotoxins. Although they belong to the 3FTx family, the characteristic functional residues of α-neurotoxins are not conserved. We evaluated the subtype specificity and structure-function relationships of Oh9-1, an Ω-neurotoxin from Ophiophagus hannah venom. Recombinant Oh9-1 showed reversible postsynaptic neurotoxicity in the micromolar range. Experiments with different nAChR subtypes expressed in Xenopus oocytes indicated Oh9-1 is selective for rat muscle type α1β1εδ (adult) and α1β1γδ (fetal) and rat neuronal α3β2 subtypes. However, Oh9-1 showed low or no affinity for other human and rat neuronal subtypes. Twelve individual alanine-scan mutants encompassing all three loops of Oh9-1 were evaluated for binding to α1β1εδ and α3β2 subtypes. Oh9-1's loop-II residues (M25, F27) were the most critical for interactions and formed the common binding core. Mutations at T23 and F26 caused a significant loss in activity at α1β1εδ receptors but had no effect on the interaction with the α3β2 subtype. Similarly, mutations at loop-II (H7, K22, H30) and -III (K45) of Oh9-1 had a distinctly different impact on its activity with these subtypes. Thus, Oh9-1 interacts with these nAChRs via distinct residues. Unlike α-neurotoxins, the tip of loop-II is not involved. We reveal a novel mode of interaction, where both sides of the β-strand of Oh9-1's loop-II interact with α1β1εδ, but only one side interacts with α3β2. Phylogenetic analysis revealed functional organization of the Ω-neurotoxins independent of α-neurotoxins. Thus, Ω-neurotoxin: Oh9-1 may be a new, structurally distinct class of 3FTxs that, like α-neurotoxins, antagonize nAChRs. However, Oh9-1 binds to the ACh binding pocket via a different set of functional residues.
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore 117543.
authors.forename: Varuna
authors.initials: V
authors.lastname: Hassan-Puttaswamy
authors.affiliation: Health Innovations Research Institute, RMIT University , Melbourne, Victoria 3083, Australia.
authors.forename: David J
authors.initials: DJ
authors.lastname: Adams
authors.affiliation: Department of Biological Sciences, National University of Singapore , Singapore 117543.
authors.forename: R Manjunatha
authors.initials: RM
authors.lastname: Kini
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D055808:Drug Discovery; D004546:Elapid Venoms / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009498:Neurotoxins / Q000737:chemistry* / Q000633:toxicity*; D010802:Phylogeny; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000378:metabolism*; D016415:Sequence Alignment; D012910:Snake Venoms / Q000737:chemistry* / Q000633:toxicity*
chemical_list: D004546:Elapid Venoms; D009498:Neurotoxins; C459589:Oh9-1 neurotoxin; D011978:Receptors, Nicotinic; D012910:Snake Venoms; D000409:Alanine