Atomic-Level Characterization of the Inhibition Mechanism of the Calcium Pump by Phospholamban

Abstract

We have performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in complex with phospholamban (PLB). Regulation of SERCA activity by PLB is central in modulating cardiac contractility. X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca2+ affinity E2 state that is incompatible with metal ion binding, thereby blocking the conversion toward a high-Ca2+ affinity E1 state. Estimation of pKa values of the acidic residues in the Ca2+ sites indicates that under normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state deprotonated at residues E309 and D800 yet protonated at residue E771. We performed microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K+ and 3 mM Mg2+. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol, and that two K+ ions bind transiently (≤5 ns), and non-specifically (9 different positions) to the two transport sites; the total occupancy time of the two K+ ions in the transport sites is 80%. These findings indicate that PLB does not inhibit the E2-to-E1 interconversion but instead populates a novel E1 intermediate, E1•H+771, that depresses SERCA activation by Ca2+. We propose that the efficient regulation of SERCA activity by PLB results from structural transitions that occur primarily in the E1 state of the pump. This work was supported by grants to L.M.E-F. from the American Heart Association (12SDG12060656) and to D.D.T from NIH (GM27906), and by the University of Minnesota Supercomputing Institute.