Thermodynamics of Cation Binding to the Sarcoendoplasmic Reticulum Calcium ATPase Pump and Impacts on Enzyme Function

Sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca²⁺) binds SERCA with micromolar affinity, magnesium (Mg²⁺) and potassium (K⁺) also compete with Ca²⁺ binding. However, the molecular bases for these competing ions' influence on the SERCA function and the selectivity of the pump for Ca²⁺ are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca²⁺ binding sites via (1) triplicate molecular dynamics (MD) simulations of Mg²⁺, Ca²⁺, and K⁺-bound SERCA, (2) mean spherical approximation (MSA) theory to score the affinity and selectivity of cation binding to the MD-resolved structures, and (3) state models of SERCA turnover informed from MSA-derived affinity data. Our key findings are that (a) coordination at sites I and II is optimized for Ca²⁺ and to a lesser extent for Mg²⁺ and K⁺, as determined by MD-derived cation-amino acid oxygen and bound water configurations, (b) the impaired coordination and high desolvation cost for Mg²⁺ precludes favorable Mg²⁺ binding relative to Ca²⁺, while K⁺ has limited capacity to bind site I, and (c) Mg²⁺ most likely acts as inhibitor and K⁺ as intermediate in SERCA's reaction cycle, based on a best-fit state model of SERCA turnover. These findings provide a quantitative basis for SERCA function that leverages molecular-scale thermodynamic data and rationalizes enzyme activity across broad ranges of K⁺, Ca²⁺, and Mg²⁺ concentrations.