Conserved region amino acid mutations in Calcin: Altering the RyR structural-functional relationship.

The scorpion venom-derived peptide Calcin is a high-affinity ligand for Ryanodine Receptors (RyRs), known to modulate calcium release by stabilizing a sub-conductance state. While previous alanine-scanning mutagenesis highlighted the importance of electrostatic interactions, the functional impact of reversing acidic residues to basic ones within its conserved regions remains unexplored. Here, we employed a combined computational and experimental approach to investigate how charge-reversal mutations at two acidic sites (E12 and E29) in OpiCa1, a potent Calcin member, affect its structure, RyR interaction, and functional efficacy. Our results demonstrated that while all mutants (E12R, E12K, E29R, E29K) maintained the native inhibitor cystine knot fold, they exhibited altered surface electrostatic potential. Molecular docking and dynamics simulations revealed distinct binding modes and stabilites with RyR1 and RyR2. Notably, the E29R mutant displayed superior performance in cellular assays, inducing a significantly stronger Ca2+ release from cardiomyocytes via RyR2 activation compared to wild-type OpiCa1 and other mutants. Our findings identify E29 as a critical residue where a charge-reversal mutation optimally enhances Calcin's activity, primarily by strengthening electrostatic complementarity with the RyR's acidic channel pore. This study provides crucial insights into the structure-function relationship of Calcin and establishes a rational basis for engineering optimized peptide therapeutics targeting RyR-related calcium dysregulation diseases.