Common Interaction Surfaces for Tarantula Toxins Targeting Kv and ASIC Channels

Abstract

Gating-modifier tarantula toxins, such as hanatoxin and GxTx-1E, have been shown to partition into membranes to interact with voltage-sensors of voltage-gated ion channels. While relatively little is known about the specific molecular interactions between these toxins and voltage sensors, PcTx1 is a related gating-modifier tarantula toxin that was recently crystallized in complex with acid sensing ion channels (ASIC); demonstrating that the toxin binds to the extracellular domain, where it interacts with helix-5 and inserts an Arg-finger into the subunit interface. Although GxTx-1E and PcTx1 interact with structurally unrelated ion channels in different environments (membrane vs solution), their structures are remarkably similar (backbone RMSD∼3 A). To compare the binding surfaces of GxTx-1E and PcTx1, we carried out Ala scanning mutagenesis on GxTx-1E and studied the interaction of each mutant with the Kv2.1 channel using electrophysiology. Our results identify an active surface of GxTx-1E that overlaps extensively with the binding surface of PcTx1 for ASIC. To further compare toxin binding surfaces, we constructed channel chimeras in which helix-5 from ASIC was transferred into Kv2.1, and a toxin chimera, in which two critical loops of GxTx-1E were transferred into PcTx1. These channel chimeras retained robust voltage-dependent activation, however they remained insensitive to PcTx1, and the toxin chimera did not inhibit Kv2.1. In addition, we used Rosetta structural modeling methods to construct docking configurations homologous to the PcTx1-ASIC structure where GxTx-1E is bound to the S3b helix of the Kv1.2/2.1 paddle chimera. Although the model has an energetically favorable peptide-protein interface, it requires that GxTx-1E span the lipid bilayer, exposing many polar residues on GxTX-1E to a hydrophobic environment. These results suggest that tarantula toxins use common surfaces to bind to structurally unrelated ion channels, yet the precise mechanisms of interaction are distinct.