Protein-Protein Interactions in Calcium Transport Regulation Probed by Saturation Transfer Electron Paramagnetic Resonance

Abstract

We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser(16). To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser(16) phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.

Allosteric regulation of SERCA by phosphorylation-mediated conformational shift of phospholamban

“…S12). Specifically, the significant change in Cα shift with respect to free PLN AFA is a strong indicator of SERCA binding and confirms that the TM domain remains bound to SERCA on phosphorylation, as recently demonstrated by EPR spectroscopy (17). When monitored by rINEPT experiments, the intensities of the 13 C resonances of PLN pS16AFA domain Ia in the free form are much higher than those of the unphosphorylated PLN AFA , a finding indicative of an order-to-disorder transition on phosphorylation (SI Appendix, Fig.…”

“…Moreover, there have been few structural data on the complex between SERCA and phosphorylated PLN. On the basis of crosslinking and sparse spectroscopic data, two different mechanistic models have been proposed: a dissociative model, in which phosphorylation causes PLN detachment from SERCA, reestablishing Ca 2+ flux (14), and the subunit model, in which phosphorylation induces conformational rearrangements of PLN's cytoplasmic domain without dissociation (15)(16)(17). Nevertheless, the two models fall short in the interpretation of the recent structure-function correlations (15,(18)(19)(20).…”

Changes in activity of hippocampus creatine kinase under circadian rhythm disorders

“…The effect is presumably caused by structural features of mitochondrial CK, particularly the existence of a sulfhydryl group in the active center which is probably not present in the cytosolic isoforms (Wyss & Kaddurah-Daouk 2000). So, the obtained results could be caused by oxidative modifications of the enzyme (James et al 2012), although a lower rate of enzyme synthesis cannot be excluded as another cause. …”

Phospholamban phosphorylation, mutation, and structural dynamics: a biophysical approach to understanding and treating cardiomyopathy

“…β-adrenergic-mediated phosphorylation of PLB at S16 by PKA, or at T17 by CaMKII, partially restores SERCA2a activity (Bers 2002; Karim et al 2006; Wegener et al 1989). Recent studies show that PLB remains bound to SERCA2a, even after phosphorylation (Dong and Thomas 2014; James et al 2012), so SERCA2a activity can be restored only by phosphorylating PLB, ablating PLB, overexpressing SERCA2a, or expressing a loss-of-inhibition mutant of PLB.…”

“…This model further validated the strategies of decreasing PLB expression and increasing PLB phosphorylation, and it motivated the search for a drug that uncouples PLB from SERCA2a (Cornea et al 2013; Ferrandi et al 2013; Huang 2013; Khan et al 2009; Rocchetti et al 2008). Recent electron paramagnetic resonance (EPR) (James et al 2012), nuclear magnetic resonance (NMR) (Gustavsson et al 2013), and fluorescence resonance energy transfer (FRET) (Dong and Thomas 2014) studies have revealed that PLB is essentially a subunit of SERCA2a, remaining tightly bound even after phosphorylation (Fig. 1B).…”