We have performed microsecond molecular dynamics (MD) simulations and protein pK(a) calculations of the muscle calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in complex with sarcolipin (SLN) to determine the mechanism by which SLN inhibits SERCA. SLN and its close analog phospholamban (PLN) are membrane proteins that regulate SERCA by inhibiting Ca2+ transport in skeletal and cardiac muscle. Although SLN and PLB binding to SERCA have different functional outcomes on the coupling efficiency of SERCA, both proteins decrease the apparent Ca2+ affinity of the pump, suggesting that SLN and PLB inhibit SERCA by using a similar mechanism. Recently, MD simulations showed that PLB inhibits SERCA by populating a metal ion-free, partially-protonated El state of the pump, E1 center dot H-771(+). X-ray crystallography studies at 40-80 mM Mg2+ have proposed that SLN-bound SERCA populates E1 center dot Mg2+, an intermediate with Mg2+ bound near transport site I. To test this proposed mode of SLN regulation, we performed a 0.5- mu s MD simulation of E1 center dot Mg2+-SLN in a solution containing 100 mM K+ and 3 mM Mg2+, with calculation of domain dynamics in the cytosolic headpiece and side-chain ionization and occupancy in the transport sites. We found that SLN increases the distance between residues E771 and D800, thereby rendering E1 center dot Mg2+ incapable of producing a competent Ca2+ transport site I. Following removal of Mg2+. a 2-mu s MD simulation of Mg2+-free SERCA-SLN showed that Mg2+ does not re-bind to the transport sites, indicating that SERCA-SLN does not populate E1 center dot Mg2+ at physiological conditions. Instead, protein pKa calculations indicate that SLN stabilizes a metal ion-free SERCA state (E1 center dot H-771(+)) protonated at residue E771, but ionized at E309 and D800. We conclude that both SLN and PLB inhibit SERCA by populating a similar metal ion-free intermediate state. We propose that (i) this partially-protonated intermediate serves as the consensus mechanism for SERCA inhibition by other members of the SERCA regulatory subunit family including myoregulin and sarcolamban, and (ii) this consensus mechanism is utilized to regulate Ca2+ transport in skeletal and cardiac muscle, with important implications for therapeutic approaches to muscle dystrophy and heart failure. (C) 2015 Elsevier Inc. All rights reserved.