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

Abstract

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.

Keywords: SERCA; calcium; calcium ATPase; cardiac muscle; inhibition mechanism; membrane transport; molecular dynamics; phospholamban; sarcoplasmic reticulum (SR).