Solution structure of Phrixotoxin 1, a specific peptide inhibitor of Kv4 potassium channels from the venom of the theraphosid spider Phrixotrichus auratus

Abstract

Animal toxins block voltage-dependent potassium channels (Kv) either by occluding the conduction pore (pore blockers) or by modifying the channel gating properties (gating modifiers). Gating modifiers of Kv channels bind to four equivalent extracellular sites near the S3 and S4 segments, close to the voltage sensor. Phrixotoxins are gating modifiers that bind preferentially to the closed state of the channel and fold into the Inhibitory Cystine Knot structural motif. We have solved the solution structure of Phrixotoxin 1, a gating modifier of Kv4 potassium channels. Analysis of the molecular surface and the electrostatic anisotropy of Phrixotoxin 1 and of other toxins acting on voltage-dependent potassium channels allowed us to propose a toxin interacting surface that encompasses both the surface from which the dipole moment emerges and a neighboring hydrophobic surface rich in aromatic residues.

Figure 1.

RP-HPLC analysis of the refolding of PaTx1. Traces obtained at different times are superimposed and offset for clarity. Timescale for trace 1 (t = 0). (Black triangle) The linear reduced form of PaTx1.

Figure 2.

(A) Sequence of PaTx1 and sequential assignments. Collected sequential nOe are classified into strong, medium, and weak nOe, and are indicated by thick, medium, and thin lines, respectively. (Arrow) The secondary elements (extended regions); (filled circles) ³J_HN-Hα coupling constants ≥ 8 Hz; (open circles) ³J_HN-Hα coupling constants ≤ 6 Hz. (B) nOe (top) and RMSD (bottom) distribution vs. sequence of PaTx1. Intraresidue nOe are in dark gray; sequential nOe, in black; medium nOe, in light gray; and long-range nOe, in white. RMSD values for backbone and all heavy atoms are in gray and in black, respectively.

Figure 3.

(A) Stereopair view of the best fit of 25 solution structures of PaTx1; the backbones of the molecules are represented. (B) MOLSCRIPT (Kraulis 1991) representation. The cystines and N and C termini are labeled. (C) Stick representation of residues Lys 4 and Trp 7. The residue Lys 4 is located in the neighborhood of Trp 7 and its amide proton, placed in the ring current of this tryptophan, has an unusual chemical shift.

Figure 4.

(A) MOLSCRIPT representation of the structure of ICK toxins that act against K_V channels. From left to right: PaTx1 and HpTx2 (Kv4.2), HaTx1 (Kv2.1), κ-PVIIA (shaker K⁺). (B) Sequence alignment of PaTx1 with other toxins acting against K⁺ channels. PaTx1, PaTx2 (Phrixotoxins 1 and 2) from Phrixotrichus auratus, HpTx2 (Heteropodatoxin 2) from Heteropoda venatoria, HaTx1 (Hanatoxin 1) from Grammostola spatulata, ScTx1 (Stromatoxin 1) from Stromatopelma calceata, HmTx1 (Heteroscodratoxin 1) from Heteroscodra maculata, PVIIA (κ-conotoxin PVIIA) from Conus purpurascens (see references in text).

Figure 5.

(A) Orientation of the dipole moment of PaTx1 (top center) emerging through Lys 26. The resulting putative functional surface of PaTx1 is represented (in the same orientation) in B, centered around Lys 26 and the hydrophobic patch (top center). The same procedure was used to generate the corresponding dipoles of HaTx1 (top left), HpTx2 (top right), ScTx1 (bottom left), and PaTx2 (bottom right). The residues are colored green for polar uncharged residues, blue for basic residues, red for acidic residues, purple for aromatic residues, and yellow for aromatic residues. (B) Putative functional surfaces of the toxins represented in CPK (TURBO software), in the same orientation as in A, centered around basic residues and the hydrophobic patch. The residues are colored green for polar uncharged residues, blue for basic residues, red for acidic residues, purple for aromatic residues, and yellow for aliphatic residues.