Role of Asn-16 and Ser-19 in Anthopleurin B Binding. Implications for the Electrostatic Nature of Na_V Site 3^,

Abstract

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.

Sea anemone venom as a source of insecticidal peptides acting on voltage-gated Na+ channels

“…Neighboring residues are either less sensitive, or their sensitivity is dependent on the nature of the mutation, especially the introduction of negative charges at these positions that is poorly tolerated. In addition, Arg 12 , Ser 19 and Lys 49 are reported to be important for toxin affinity and channel isoform specificity of AP-B (Gallagher and Blumenthal, 1994;Seibert et al, 2004). It should be noted that AP-B has no selectivity between neuronal and cardiac Na v channels, while AP-A is selective for the latter.…”

Structural Basis for the Voltage-gated Na+ Channel Selectivity of the Scorpion α-Like Toxin BmK M1

“…Position 8 in the native sequence of BmK M1 is occupied by a long-chain basic residue, lysine, which protrudes from the five-residue reverse turn (8)(9)(10)(11)(12) and is fully exposed to the solvent. The flanking peptide bond (9-10) adopts an unusual cis form ( Figure 5).…”

“…7 The scorpion a-toxins, funnel web spider toxins and the sea anemone a-toxins all bind to site 3, resulting in delayed inactivation of the channel. [8][9][10][11] Here, we will focus on a toxin that interacts with this site. According to their different pharmacological preferences, scorpion a-toxins can be divided into three subgroups: classical a; a-like; and insect a-toxins.…”

“…17,[19][20][21][22][23][24][25] Previous studies revealed three functionally important epitopes that underlie the molecular basis of mammal and/or insect-selectivity: 24,25 (1) the first three N-terminal residues; (2) the five-residue reverse turn (residues [8][9][10][11][12] in combination with the C-tail (residues 57-64); and (3) the loop between the b 1 and b 2 sheet (residues [40][41][42][43]. The region consisting of the reverse turn (residues [8][9][10][11][12] in combination with the C-tail (57-64), previously termed "Site RC", 17 has received much attention due to its unique tertiary arrangement with an unusual non-proline cis peptide bond between residues 9 and 10. 17,26 Mutagenesis analysis revealed that this site is involved in the binding preference for phylogenetically distinct VGSCs.…”

“…As a result, 11 mutants of residues in the five-residue reverse turn (8)(9)(10)(11)(12) and C-tail (57-64) were thoroughly analysed. We found a molecular switch at position 8 (K8), where the substitution with specific residues can redirect the a-like characteristics of BmK M1 to either insect specificity or much higher mammalian specificity.…”