Structural basis for relief of the sarcoplasmic reticulum Ca ²⁺ -ATPase inhibition by phospholamban at saturating Ca ²⁺ conditions

We have performed extensive atomistic molecular dynamics simulations to probe the structural mechanism for relief of sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) inhibition by phospholamban (PLB) at saturating Ca 2+ conditions. Reversal of SERCA-PLB inhibition by saturating Ca 2+ operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Simulation of the inhibitory complex at super-physiological Ca 2+ concentrations ([Ca 2+ ]=10 mM) revealed that calcium ions interact primarily with SERCA and the lipid headgroups, but not with the cytosolic domain of PLB or the cytosolic side of the SERCA-PLB interface. At this [Ca 2+ ], a single Ca 2+ ion is translocated from the cytosol to the transmembrane transport sites. We used this Ca 2+ -bound complex as an initial structure to simulate the effects of saturating Ca 2+ at physiological conditions ([Ca 2+ ] total≈ 400 μM). At these conditions, ~30% of the Ca 2+ -bound complexes exhibit structural features that correspond to an inhibited state. However, in ~70% of the Ca 2+ -bound complexes, Ca 2+ moves to transport site I, recruits Glu771 and Asp800, and disrupts key inhibitory contacts involving conserved PLB residue Asn34. Structural analysis showed that Ca 2+ induces only local changes in interresidue inhibitory interactions, but does not induce dissociation, repositioning or changes in the structural dynamics of PLB. Upon relief of SERCA inhibition, Ca 2+ binding produces a productive site I configuration that is sufficient for subsequent SERCA activation. We propose that at saturating [Ca 2+ ] and in the absence of PLB phosphorylation, binding of a single Ca 2+ ion in the transport sites rapidly shifts the equilibrium toward a non-inhibited SERCA-PLB complex.