A Small Loop in the SERCA N-Domain Facilitates the Transition to a Compact Conformation

Abstract

The cardiac sarco/endoplasmic reticulum (SR) calcium ATPase (SERCA) and its inhibitor phospholamban (PLB) are key regulators of calcium levels in cardiac cells. SERCA contributes to calcium re-uptake from the cytosol into the SR, resulting in the relaxation of the muscles. PLB phosphorylation leads to SERCA relief-of-inhibition, and subsequently improved contractility and faster relaxation due to increased SR calcium load. The current project is aimed at revealing the details of the SERCA transition from an open to a closed conformation. In addition, the experiments provide the insight into the mechanism of the SERCA relief of inhibition after PLB phosphorylation. Molecular dynamics (MD) simulations led us to hypothesize that H-bonds between a small loop in SERCA N-domain and the A-domain facilitate SERCA headpiece closure. We propose that the cytoplasmic domain of phosphorylated PLB facilitates the transition to a closed conformation by interaction with this discrete loop of SERCA N-domain. Other groups had reported that mutation of N-loop residues reduces ATPase activity by 35-60%. Using fluorescence resonance energy transfer (FRET) experiments and MD simulations, we find that SERCA N-loop mutations impair the closure of cytoplasmic headpiece. Furthermore, SERCA precipitation with GST-PLB is reduced upon N-loop mutation. MD simulations of a SERCA-PLB docking model suggest that PLB phosphorylation facilitates an interaction of the PLB cytoplasmic domain with SERCA A-domain and the N-domain loop, leading to a more compact conformation of the SERCA headpiece. Indeed, we observe a change in FRET between SERCA and PLB after PLB phosphorylation, consistent with a conformational change of the SERCA-PLB complex. Together, our data implicate a small N-domain loop in SERCA transitioning to a compact conformation. This discrete loop might potentially become a new target for rational design of novel therapeutics to modulate SERCA activity.