Alanine Mutagenesis Identifies Specific Amino Acids of Nemertide Alpha-1 Activity and Its Binding to Target Receptors

We recently discovered and characterized a novel family of peptide toxins, the alpha-nemertides, from the marine ribbon worm <i>Lineus longissimus</i>. These 31-residue peptides show potent neurotoxicity against invertebrate voltage-gated sodium (Na_v) channels, making them promising candidates for biopesticide development. To explore structure-activity relationships, we synthesized 20 nemertide alpha-1 mutants (17 alanine, 3 lysine substitutions) to identify residues critical for activity and selectivity. Key positions, including S12, T17, N19, W22, and F24, were found to influence activity on Na_v channels significantly. Notably, the S12A mutant showed high selectivity for invertebrate Na_vs, suggesting its potential as a selective tool or lead scaffold. Our findings highlight critical interaction points likely to be involved in binding to site 3 of domain IV on Na_v channels and demonstrate how targeted modifications can sharpen selectivity. These insights support the rational design of more selective peptides and identify S12A as a promising candidate for further development as a biopesticide.