Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban

Abstract

We have used site-directed spin labeling and electron paramagnetic resonance (EPR) to map interactions between the transmembrane (TM) domains of the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) as affected by PLB phosphorylation. In the cardiac sarcoplasmic reticulum, PLB binding to SERCA results in Ca-dependent enzyme inhibition, which is reversed by PLB phosphorylation at Ser16. Previous spectroscopic studies on SERCA-PLB have largely focused on the cytoplasmic domain of PLB, showing that phosphorylation induces a structural shift in this domain relative to SERCA. However, SERCA inhibition is due entirely to TM domain interactions. Therefore, we focus here on PLB's TM domain, attaching Cys-reactive spin labels at five different positions. In each case, continuous-wave EPR indicated moderate spin-label mobility, with the addition of SERCA revealing two populations, one indistinguishable from PLB alone and another with more restricted rotational mobility, presumably due to SERCA-binding. Phosphorylation had no effect on the rotational mobility of either component but significantly decreased the mole fraction of the restricted component. Solvent-accessibility experiments using power-saturation EPR and saturation-recovery EPR confirmed that these two spectral components were SERCA-bound and unbound PLB and showed that phosphorylation increased the overall lipid accessibility of the TM domain by increasing the fraction of unbound PLB. However-based on these results-at physiological levels of SERCA and PLB, most SERCA would have bound PLB even after phosphorylation. Additionally, no structural shift in the TM domain of SERCA-bound PLB was detected, as there were no significant changes in membrane insertion depth or its accessibility. Therefore, we conclude that under physiological conditions, the phosphorylation of PLB induces little or no change in the interaction of the TM domain with SERCA, so relief of inhibition is predominantly due to the previously observed structural shift in the cytoplasmic domain.

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

“…S1-S9, Supporting information). These findings agree with previous studies showing that binding of a single Ca 2+ ion relieves inhibitory SERCA-PLB contacts without inducing changes in the overall architecture of the SERCA-PLB interface [17] or secondary structure of PLB, such as those induced by PLB phosphorylation [13][14][15][16]27].…”

Atomistic Structure and Dynamics of the Ca2+-ATPase Bound to Phosphorylated Phospholamban

“…Furthermore, it is known that the cytosolic domain of PLB becomes structurally disordered upon phosphorylation, yet there has been speculation that SERCA has a well-defined pocket that binds this unstructured domain, and that this interaction is directly responsible for relief of SERCA inhibition [ 15 ]. More recent studies have shown that phosphorylation of PLB does not induce substantial changes in the interaction of the inhibitory transmembrane (TM) domain of PLB [ 16 ], thus suggesting that SERCA regulation by PLB phosphorylation is likely due to an allosteric effect exerted by the disordered cytosolic domain of PLB.…”

Protein Docking and Steered Molecular Dynamics Reveal Alternative Regulatory Sites on the SERCA Calcium Transporter

“…Some studies suggested that SERCA inhibition is relieved by dissociation of the complex (10)(11)(12). However, previous studies by our lab (13)(14)(15)(16) and others (17)(18)(19)(20)(21)(22)(23) indicate that PLB remains bound after phosphorylation and in high Ca, and relief of inhibition is due to a conformational change of the intact regulatory complex. Ca-dependent conformational changes in the SERCA-PLB complex are likely to include alterations in the canonical PLB-binding site on SERCA, a cleft composed of transmembrane helices M2, M6, and M9.…”