Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3.
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.
Seibert
Anna L
AL
Department of Medicine, University of Chicago, Chicago, Illinois 60637, USA.
Liu
Jinrong
J
Hanck
Dorothy A
DA
Blumenthal
Kenneth M
KM
D019943:Amino Acid Substitution / Q000235:genetics*; D000818:Animals; D001216:Asparagine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002478:Cells, Cultured; D003064:Cnidarian Venoms / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004594:Electrophysiology; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000378:metabolism*; D011485:Protein Binding; D012615:Sea Anemones / Q000737:chemistry; D012694:Serine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015222:Sodium Channels / Q000378:metabolism*; D055672:Static Electricity
D003064:Cnidarian Venoms; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D015222:Sodium Channels; D012694:Serine; D001216:Asparagine; C026132:anthopleurin B