Calmodulin Interaction Interface with Plasma Membrane Ca²⁺-ATPase Isoforms: An Integrative Bioinformatic Analysis

Plasma membrane Ca²⁺-ATPases (PMCA) are activated by calmodulin (CaM) via a C-terminal calmodulin-binding domain, CaMBD. Although specific mutations in this domain have been linked to disease, the broader impact of alternative substitutions across the interface remains unexplored. We applied an integrative in silico workflow to test six substitutions within CaMBD positions 1-18, L5R, N6I, I8T, V14E/D, and F18S, across PMCA isoforms 1-4. CaMBD sequences were aligned across isoforms, and candidates for substitutions were selected by conservation and nucleotide feasibility, prioritizing conserved or co-evolutionarily relevant sites, with substitutions possible by single-nucleotide change. PolyPhen-2 screened the impact of the substitutions on the protein functionality, the DisGeNET database was used to contextualize ATP2B genes with clinical phenotypes, and structural models plus binding free energy changes were estimated with AlphaFold3, FoldX, and MutaBind2. Effects were isoform and subregion dependent, with the strongest weakening toward the CaMBD C-terminus. V14E/D and F18S showed the largest and consistent predicted destabilization, consistent with disruption of conserved hydrophobic anchors. I8T and L5R had mixed outcomes depending on isoform, while N6I presented various scenarios with no clear effect. PolyPhen-2 classified most tested substitutions as damaging. Gene-disease evidence linked ATP2B to neurological, endocrine, and oncologic phenotypes, consistent with roles in Ca²⁺ homeostasis. Overall, CaMBD appears highly sensitive to perturbation, with distal positions 14-18 particularly vulnerable to substitutions that can destabilize CaM binding and potentially impair PMCA-mediated Ca²⁺ clearance in susceptible tissues.