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

Area of Science:

• Biochemistry and Molecular Biology
• Structural Biology
• Computational Biology

Background:

• Plasma membrane Ca2+-ATPases (PMCA) regulate cellular calcium homeostasis.
• Calmodulin (CaM) activates PMCA through its C-terminal calmodulin-binding domain (CaMBD).
• Specific CaMBD mutations are linked to diseases, but the impact of other substitutions is largely unknown.

Purpose of the Study:

• To investigate the functional impact of six specific substitutions within the PMCA CaMBD.
• To assess isoform-specific effects and identify vulnerable CaMBD regions.
• To correlate predicted functional changes with known gene-disease associations for ATP2B genes.

Main Methods:

• An integrative in silico workflow combining sequence alignment, conservation analysis, and nucleotide feasibility.
• Protein function prediction using PolyPhen-2.
• Gene-disease association analysis using the DisGeNET database.
• Structural modeling and binding free energy estimation using AlphaFold3, FoldX, and MutaBind2.

Main Results:

• Substitutions V14E/D and F18S showed the most significant predicted destabilization of CaMBD, particularly at the C-terminus.
• Effects were isoform-dependent, with some substitutions (I8T, L5R) yielding mixed outcomes.
• Most tested substitutions were predicted as damaging by PolyPhen-2, and ATP2B gene associations linked them to neurological, endocrine, and oncologic conditions.

Conclusions:

• The PMCA CaMBD is highly sensitive to amino acid substitutions.
• Positions 14-18 in the CaMBD are particularly vulnerable, and destabilizing mutations may impair CaM binding and PMCA function.
• These findings highlight the potential for CaMBD variants to contribute to human diseases through disrupted calcium homeostasis.