Contribution of cationic and aromatic residues to activity of a unique neurotoxin, Cerebratulus lacteus B-IV.

摘要

Cerebratulus lacteus toxin B-IV represents a novel class of sodium channel modulators with unique structure and species selectivity. This 55-residue, highly cationic polypeptide contains two antiparallel {dollar}alpha{dollar}-helices which together include more than 50% of the entire sequence. It delays the inactivation process of crustacean sodium channels upon binding to a single class of receptor sites, similar to the actions of Catterall's site III toxins. Site-directed mutagenesis has been utilized to assess the functional contributions of individual amino acid residues in different regions of this toxin. Within the N-terminal helix, while lysine, glutamine and alanine substitutions of Arg-17 result in complete ablation of B-IV toxicity, charge neutralizing mutations of Lys-18 and Lys-19 as well as the two carboxylates Glu-13 and Asp-21 have little effect on the biological activity. These Arg-17 muteins have similar secondary structures and thermal stabilities compared to that of the wild-type toxin as judged by circular dichroism spectroscopy. suggesting a primary role played by the Arg-17 guanidinium group in channel binding. On the other hand, in the multi-turn loop region linking the two helices, charge neutralizing replacement of Arg-25 by glutamine causes a 400-fold reduction in specific toxicity while the charge conserving mutein R25K has very similar activity as the natural toxin. These observations therefore demonstrate the requirement for a positive charge at position 25. In the same region, replacement of Trp-30 with a serine results in loss of more than 40-fold activity whereas substitution with a tyrosine recovers to a near wild-type value, indicating that the aromatic side chain at this position is required for toxin function. Charge neutralizing substitution of Lys-29 or Lys-33 has very little effect on toxicity, suggesting the non-essentiality of these residues. Analogous studies of the third arginine, Arg-34, which is located in the beginning of the C-terminal helix, it plays the smallest role in activity in that its replacement by glutamine and alanine diminishes toxicity by only 20- and 70-fold, respectively. However, thermal denaturation experiments suggest that this arginine may be an important for in B-IV stability.