Surface Characterization and Membrane Interaction of Double-Knot Toxin, an Activator of TRPV1 Channels

Double-knot toxin (DkTx) is a novel tarantula toxin that activates TRPV1 channels by binding to the extracellular pore domain of the channel, and is composed of two lobes named knot1(K1) and knot2(K2). Previous studies have shown that both lobes can be synthesized separately and activate the channel with different affinities. Recently, near atomic resolution structures of TRPV1 in distinct states (apo, capsaicin bound and DkTx&RTx bound) were reported using electron cryo-microscopy. These structures show that TRPV1 adopts a structure that is similar to Kv channels, and that the pore domain undergoes distributed conformational change upon activation in response to binding of DkTx and RTx. Although these structures show where DkTx binds, they do not have sufficient resolution to reveal the structural basis of the toxin-channel interaction. Here we solved the solution NMR structure of DkTx and dock that structure into the DkTx/RTx bound electron density maps using the Xplor-NIH program. Our results show that the toxin binds to a perimeter of the pore domain at the interface between the pore helix and S6 of neighboring subunit of the channel, and demonstrate that the toxin-channel interface is dominated by hydrophobic interactions. Interestingly, when bound to the channel, several residues on the toxin extend over the edge of the pore domain where they would be expected in interact with the surrounding membrane. To explore this possibility we tested whether DkTx can interact with membranes using a tryptophan fluorescence assay (each lobe contains a single conserved tryptophan). Indeed, each lobe of DkTx interacts with membranes, and the interaction is energetically more favorable in the bivalent toxin.