Abstract
Sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) is a transmembrane pump that plays an important role in transporting calcium into the sarcoplasmic reticulum (SR). While calcium (Ca(2+)) binds SERCA with micromolar affinity, magnesium (Mg(2+)) and potassium (K(+)) also compete with Ca(2+) binding. However, the molecular bases for these competing ions' influence on the SERCA function and the selectivity of the pump for Ca(2+) are not well-established. We therefore used in silico methods to resolve molecular determinants of cation binding in the canonical site I and II Ca(2+) binding sites via (1) triplicate molecular dynamics (MD) simulations of Mg(2+), Ca(2+), 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 Ca(2+) and to a lesser extent for Mg(2+) and K(+), as determined by MD-derived cation-amino acid oxygen and bound water configurations, (b) the impaired coordination and high desolvation cost for Mg(2+) precludes favorable Mg(2+) binding relative to Ca(2+), while K(+) has limited capacity to bind site I, and (c) Mg(2+) 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(+), Ca(2+), and Mg(2+) concentrations.