Dynamics-driven allostery underlies pre-activation of the regulatory Ca2+-ATPase/phospholamban complex

Abstract

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+-dependent activation of SERCA-PLB provides a rheostat function that regulates cytosolic and SR Ca2+ levels. While experimental and computational studies alone have led to a greater insight into the mechanisms for SERCA-PLB regulation, the structural changes induced by Ca2+ binding and how those are communicated to couple enzymatic activity with active transport remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 s and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to Ca2+ binding. Complementary simulations and experiments showed structural disorder underlies PLB inhibition of SERCA, and Ca2+ binding is sufficient to shift the protein population toward a structurally ordered state of the complex. This structural transition results in a redistribution of structural states toward a partially closed conformation of SERCAs cytosolic headpiece. Closure is accompanied by functional interactions between the N-domain {beta}5-{beta}6 loop and the A-domain. Regulation of these key structural elements indicate that Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that pre-activate SERCA-PLB. These findings provide direct support that dynamically driven protein allostery underlies PLB regulation of SERCA. These functional insights at unprecedented spatiotemporal resolution suggest a general modular architecture mechanism for dynamic regulation of the SERCA-PLB complex. Understanding these mechanisms is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases.