The heteronemertine worm Cerebratulus lacteus produces a family of four homologous polypeptide neurotoxins which alter the gating kinetics of Na- channels in crustacean neurons but are without effect on nerves from other animals. The molecular mechanism of action of these B-toxins, and the relationship between their binding site and that for other axonal toxins will be assessed. We have cloned a synthetic gene which encodes toxin B-IV, the most abundant of the B-variants, and expressed this gene in high yield in E. coli as a fusion protein from which fully active B-IV has been cleaved, laying the groundwork for the experiments to be carried out in this proposal. Regions of sequence and specific amino acid residues involved in defining interactions between B toxin and receptor will be probed by site-directed mutagenesis; a number of mutant genes have already been constructed, expressed and their protein products assayed for toxicity. The choice of future sites and substitutions is based upon the results of previous chemical modification studies carried out in this lab and on considerations of variation in specific toxicity among the B-homologs. Point mutagenesis will focus on groups of residues (e.g., Arg) previously implicated as important for activity; this technique will also allow us to assess the importance of secondary structural motifs in biological activity. The conformation of all mutants will be assessed by spectropolarimetry and conformational stabilities measured by titration of the CD spectrum as a function of denaturant concentration. In collaborative efforts, we will determine the solution structure of B-IV by 2D-NMR and also its crystal structure. These structures will be of significant value in design of future mutagenesis experiments. 2D-NMR will also be used to analyze the conformation of functionally interesting mutant forms. Identification of the channel subunit to which toxin B-IV binds will be reevaluated. Completion of the experiments proposed in this application will allow us to precisely define the nature of the contacts between this toxin and regions of the voltage-sensitive Na channel which are coupled to and important for the gating mechanism.
