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

Sarcoplasmic reticulum Ca-ATPase (SERCA) is critical for cardiac Ca transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca 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 Ca concentrations ([Ca] = 10 mm) revealed that Ca 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 [Ca], a single Ca ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca-bound complex as an initial structure to simulate the effects of saturating Ca at physiological conditions ([Ca] ≈ 400 μm). At these conditions, ∼30% of the Ca-bound complexes exhibited structural features consistent with an inhibited state. However, in ∼70% of the Ca-bound complexes, Ca moved to transport site I, recruited Glu and Asp, and disrupted key inhibitory contacts involving the conserved PLB residue Asn Structural analysis showed that Ca induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca] and in the absence of PLB phosphorylation, binding of a single Ca ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA-PLB complex.