Structure-Activity Relationship Studies Reveal that the Spider Toxin Protx-II has Unusual Membrane-Binding Properties and Inhibits NAV1.7 Channel at the Membrane Surface

Abstract

ProTx-II, a peptide toxin isolated from spider venom, has large potential as a pain therapeutic. This disulfide-rich peptide inhibits the membrane voltage-gated sodium channel Nav 1.7, a recognized therapeutic target to treat pain, through binding to the voltage-sensor domains of the channel inserted in the membrane. The exact peptide-channel binding site and the importance of the membrane in its inhibition activity remain unknown. In the current study we examined the structure and the membrane-binding properties of ProTx-II, and of a series of analogues, using NMR spectroscopy, surface plasmon resonance and fluorescence spectroscopy methodologies. Our studies reveal that ProTx-II has an amphipathic surface and high affinity for lipid bilayers with preference for negatively-charged lipids. Interestingly, despite of each of the four Trp residues being essential for its membrane-binding properties, the peptide has a shallow location in the bilayer. Furthermore, contrary to other membrane-active peptides, replacement of Lys with Arg residues decreases the membrane-binding properties of ProTx-II. Our studies also suggest that sphingomyelinase D, an enzyme present in spider venoms that converts the neutral sphingomyelin into a negatively-charged lipid, increases the ability of ProTx-II to bind to membranes that mimic raft-like domains where the ion channels are located. Overall these results suggest: 1) ProTx-II interacts with Nav 1.7 voltage sensor domain at the membrane surface; 2) sphingomyelinase D in spider venoms increases the concentration of positively-charged toxins in the channel vicinity by converting the neutral raft-domains into negatively-charged surfaces.