NMR Analysis of Interaction of LqhαIT Scorpion Toxin with a Peptide Corresponding to the D4/S3−S4 Loop of Insect Para Voltage-Gated Sodium Channel

Abstract

Voltage-gated sodium channels (Navs) are large transmembrane proteins that initiate action potential in electrically excitable cells. This central role in the nervous system has made them a primary target for a large number of neurotoxins. Scorpion alpha-neurotoxins bind to Navs with high affinity and slow their inactivation, causing a prolonged action potential. Despite the similarity in their mode of action and three-dimensional structure, alpha-toxins exhibit great variations in selectivity toward insect and mammalian Navs, suggesting differences in the binding surfaces of the toxins and the channels. The scorpion alpha-toxin binding site, termed neurotoxin receptor site 3, has been shown to involve the extracellular S3-S4 loop in domain 4 of the alpha-subunit of voltage-gated sodium channels (D4/S3-S4). In this study, the binding site for peptides corresponding to the D4/S3-S4 loop of the para insect Nav was mapped on the highly insecticidal alpha-neurotoxin, LqhalphaIT, from the scorpion Leiurus quinquestriatus hebraeus, by following changes in the toxin amide 1H and 15N chemical shifts upon binding. This analysis suggests that the five-residue turn (residues LqK8-LqC12) of LqhalphaIT and those residues in its vicinity interact with the D4/S3-S4 loop of Nav. Residues LqR18, LqW38, and LqA39 could also form a patch contributing to the interaction with D4/S3-S4. Moreover, a new bioactive residue, LqV13, was identified as being important for Nav binding and specifically for the interaction with the D4/S3-S4 loop. The contribution of LqV13 to NaV binding was further verified by mutagenesis. Future studies involving other extracellular regions of Navs are required for further characterization of the structure of the LqhalphaIT-Navs binding site.

Evolutionary Diversification of Mesobuthus α-Scorpion Toxins Affecting Sodium Channels

“…Our analysis identified six new PSSs, in which five (8, 9, 15, 38, and 50) fall into the functional region. The first finding that sites 8 and 9 undergoing positive selection is of particular interest because in Lqh␣IT, these two sites were found to be directly involved in binding to the loop connecting S3-S4 of DIV (abbreviated as L DIV:S3-S4 ) (59) whereas in BmKM1, the residue couplet (8, 9) has been proposed to act as a molecular switch where the substitution at site 8 with specific residue can redirect the ␣-like characteristics of BmKM1 to either insect or much higher specificity to (Fig. 11).…”

“…To observe the locations of these three loops on the channel, we reconstructed a model structure of DmNa v 1, in which L DI:S5-S6 was found to be located between L DIV:S5-S6 and L DIV:S3-S4 , making these three loops form a contiguous region (Fig. 12A and 12B) (59). Of these functional residues, three (8,9, and 18) have been identified here as PSSs, suggesting that this loop constitutes a critical driving force for the adaptive evolution of Mesobuthus ␣-scorpion toxins.…”

Molecular Requirements for Recognition of Brain Voltage-gated Sodium Channels by Scorpion α-Toxins

“…Subtle Alterations at the Bioactive Surface Lead to Marked Differences in Toxin Selectivity-As previously mentioned, a rational approach in the design of a target-selective scorpion toxin first requires that amino acid residues involved in bioac- (21,22,51). Note the division of the bioactive surface in the three toxins to a core domain and an NC domain.…”

Site-directed mutagenesis of the toxin from the Chinese scorpion Buthus martensii Karsch (BmKAS): Insight into sites related to analgesic activity

“…Recently, Schnur et al (2008) has shown that peptides corresponding in sequence to the extracellular S3-S4 loop in domain 4 of the α-subunit of Na v s (D4/S3-S4), exhibit millimolar affinities for LqhαIT. This provided new insights into receptor site 3 and its toxin binding mechanism.…”