Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca2+-ATPase at saturating Ca2+ conditions.

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is critical for cardiac Ca2+ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca2+ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca2+ concentrations ([Ca2+] = 10 mm) revealed that Ca2+ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA-PLB interface. At this [Ca2+], a single Ca2+ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total ≈ 400 μm). At these conditions, ∼30% of the Ca2+-bound complexes exhibited structural features consistent with an inhibited state. However, in ∼70% of the Ca2+-bound complexes, Ca2+ moved to transport site I, recruited Glu771 and Asp800, and disrupted key inhibitory contacts involving the conserved PLB residue Asn34 Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca2+ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex.