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.

Understanding Ion Binding Affinity and Selectivity in ß-Parvalbumin Using Molecular Dynamics and Mean Spherical Approximation Theory.

Parvalbumin (PV) is a globular calcium (Ca(2+))-selective protein expressed in a variety of biological tissues. Our computational studies of the rat ß-parvalbumin (ß-PV) isoform seek to elucidate the molecular thermodynamics of Ca(2+) versus magnesium (Mg(2+)) binding at the protein's two EF-hand motifs. Specifically, we have utilized molecular dynamics (MD) simulations and a mean-field electrolyte model (mean spherical approximation (MSA) theory) to delineate how the EF-hand scaffold controls the "local" thermodynamics of Ca(2+) binding selectivity over Mg(2+). Our MD simulations provide the probability density of metal-chelating oxygens within the EF-hand scaffolds for both Ca(2+) and Mg(2+), as well the conformational strain induced by Mg(2+) relative to Ca(2+) binding. MSA theory utilizes the binding domain oxygen and charge distributions to predict the chemical potential of ion binding, as well as their corresponding concentrations within the binding domain. We find that the electrostatic and steric contributions toward ion binding were similar for Mg(2+) and Ca(2+), yet the latter was 5.5 kcal/mol lower in enthalpy when internal strain within the EF hand was considered. We therefore speculate that beyond differences in dehydration energies for the Ca(2+) versus Mg(2+), strain induced in the ß-PV EF hand by cation binding significantly contributes to the nearly 10,000-fold difference in binding affinity reported in the literature. We further complemented our analyses of local factors governing cation binding selectivity with whole-protein (global) contributions, such as interhelical residue-residue contacts and solvent exposure of hydrophobic surface. These contributions were found to be comparable for both Ca(2+)- and Mg(2+)-bound ß-PV, which may implicate local factors, EF-hand strain, and dehydration, in providing the primary means of selectivity. We anticipate these methods could be used to estimate metal binding thermodynamics across a broad range of PV sequence homologues and EF-hand-containing, Ca(2+) binding proteins.