Structure and Orientation of a Voltage-Sensor Toxin in Lipid Membranes

Abstract

Amphipathic protein toxins from tarantula venom inhibit voltage-activated potassium (Kv) channels by binding to a critical helix-turn-helix motif termed the voltage sensor paddle. Although these toxins partition into membranes to bind the paddle motif, their structure and orientation within the membrane are unknown. We investigated the interaction of a tarantula toxin named SGTx with membranes using both fluorescence and NMR spectroscopy. Depth-dependent fluorescence-quenching experiments with brominated lipids suggest that Trp30 in SGTx is positioned approximately 9 A from the center of the bilayer. NMR spectra reveal that the inhibitor cystine knot structure of the toxin does not radically change upon membrane partitioning. Transferred cross-saturation NMR experiments indicate that the toxin's hydrophobic protrusion contacts the hydrophobic core of the membrane, whereas most surrounding polar residues remain at interfacial regions of the bilayer. The inferred orientation of the toxin reveals a twofold symmetry in the arrangement of basic and hydrophobic residues, a feature that is conserved among tarantula toxins. These results have important implications for regions of the toxin involved in recognizing membranes and voltage-sensor paddles, and for the mechanisms by which tarantula toxins alter the activity of different types of ion channels.

Structural interactions of a voltage sensor toxin with lipid membranes

“…Quenching was most pronounced when examining lipids with bromine attached near the middle of the acyl chain (9,10-diBr), and analysis of the quenching profile yielded a value of 9 Å from the center of the bilayer (Fig. 1F), consistent with what has been reported for related tarantula toxins hanatoxin and SGTx1 (3,44,57). These data do not provide a reliable measure of the depth of tarantula toxin partitioning into membranes (58) because there is substantial thermal disorder of both the quenching lipids and of the fluorophore, deformations of the membrane around the toxin, as well as the presence of three Trp residues in VSTx1.…”

Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNa V 1.7

“…Briefly, electrostatic interactions between gHwTx-IV and the membrane would attract the peptide to the membrane surface and additional hydrogen bonding and hydrophobic interactions would orient the peptide such that the face containing R26, K27 and K32 is available for interactions with hNa V 1.7 ( Figure 6). In addition, the overall decrease in anionic charge and increase in hydrophobicity of gHwTx-IV might further augment interactions between the peptide and hNa V 1.7 This hypothesis was previously proposed by Revell and colleagues [13], when the authors observed an increase in the inhibitory potency of the analogue [E1G,E4G,Y33W]HwTx-IV [21]. In silico docking studies suggested that the residues F6 and Y33 form hydrophobic interactions with M750 on hNa V 1.7; thus, a supporting explanation for the increased activity observed with gHwTx-IV is that the mutations F6W and…”

“…This structural feature has been proposed to promote not only GMT-voltage-gated ion channel interactions, but also GMT-lipid membrane interactions [12,13,17,18,48]. Several spider GMTs have been shown to interact with model membranes and the unique structures of these peptides have been proposed to facilitate a tri-molecular interaction involving the GMTs, the lipid membrane and target voltage-gated ion channels [14,47,[50][51][52].…”

“…Structure activity relationship studies between the toxins and their target channels reveal that this surface motif appears to be involved in the binding of GMTs to target residues on the voltage-gated ion channels and that these peptides recognize overlapping target residues on the channels [12][13][14]. For example, the channel binding region of ProTx-II has been determined to bind to F813, D816 and E818 on hNa V 1.7, whereas HwTx-IV has been shown to interact with E811, L814, D816, E818 on the same channel [15,16].…”

Rational Engineering Defines a Molecular Switch That Is Essential for Activity of Spider-Venom Peptides against the Analgesics Target NaV1.7

“…together, the protein and lipid interactions prevent the voltage sensor from making the outward movement needed for channel activation (Lee and MacKinnon, 2004;Milescu et al, 2007Milescu et al, , 2009Jung et al, 2010) Furthermore, it has been demonstrated that Hh2a is able to bind to the closed state of Na V 1.7 (Xiao et al, 2008), which agrees well with models proposing that the peptide reaches the voltage sensor while it is still embedded in the membrane, thereby blocking the outward movement of the sensor during channel activation (Henrion et al, 2012). These studies suggest that lipid interactions can be directly involved in the mechanism of channel inhibition by venom peptides.…”