Structural Characterization of Double-Knot Toxin, an Activator of TRPV1 Channels

Abstract

Venom from poisonous organisms is a rich source of peptide toxins interacting with different ion channels proteins. These peptide toxins modulate ion channels by different mechanisms, and have been widely used as tools for investigating ion channel mechanisms. Double-knot toxin (DkTx) is a novel peptide toxin that activates TRPV1 channels, and contains two inhibitory cysteine knot (ICK) motifs, as its name suggests. Previous studies show that DkTx activates TRPV1 channels, and suggest that the avidity of the toxin (slow unbinding) arises from its bivalent nature. Here we use solid-phase peptide synthesis to individually produce the two knots of DkTx (K1 and K2), fold each in vitro, and find that they exhibit different binding affinities for the channel even though they share high sequence homology. As a first step toward understanding the structural and functional relationship of DkTx binding to TRPV1 channels, we determined solution structures of each knot in using NMR. The structures show that DkTx is composed of two notably amphipathic ICK motifs (each with two beta-strands) that are connected by a flexible linker, and that K2 has a larger hydrophobic surface compared to K1. In addition, the single conserved Trp residue in each knot show different orientations, with that in K1 exhibiting greater solvent exposure. Interestingly, using intrinsic Trp fluorescence, we observe strong partitioning of DkTx and K1, but see no evidence of membrane partitioning for K2. We also made a series of K1/K2 chimeras, and identified variant residues in two loops and the C-terminus that are responsible for the higher activity of K2. From these results we propose that membrane interactions are involved in the mechanisms of DkTx activation of TRPV1, and identify surfaces of the two knots that likely involved in binding to the channel.