Phosphomimetic Mutations Increase Phospholamban Oligomerization and Alter the Structure of Its Regulatory Complex

Abstract

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA؉CaMKII sites). FRET increased with protein concentration up to a maximum (FRET max ) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K D ) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET max was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.Phospholamban (PLB) 2 is a 52-amino acid peptide localized primarily to the sarcoplasmic reticulum of cardiac muscle cells. The peptide comprises two ␣-helical domains connected by a flexible linker. NMR studies suggest an L-shaped tertiary structure; the PLB cytoplasmic domain N-terminal ␣-helix (domain IA) is significantly parallel to the bilayer surface, while the transmembrane helix (domain II) is roughly normal to the membrane (1, 2). The monomeric form of PLB binds and inhibits the sarco(endo)plasmic reticulum calcium ATPase (SERCA) (3). Inhibition is partially relieved by phosphorylation of PLB by PKA (4) and CaMKII (5), and PLB is a major effector of adrenergic signaling (6). PLB also binds to itself, forming pentamers (7). Most evidence suggests that the pentamer is unable to bind or regulate SERCA (8, 9). NMR and EPR (1) and FRET (10, 11) data suggest the pentamer is a "pinwheel" quaternary complex of L-shaped subunits, however, other diverse structures have been proposed (12)(13)(14).Phosphorylation has been proposed to alter the architecture of PLB. Wegener and Jones (7) observed a significant shift in the PLB pentamer electrophoretic mobility in SDS gels, and postulated that mobility was changed as a result of altered quaternary conformation of the pentamer. Specifically, they envisioned PLB cytoplasmic domains assuming a more compact conformation after phosphorylation (15). Consistent with such a structural transition is a recent NMR study showing that phosphorylation decreases the membrane association of the phospholamban cytoplasmic domain (16). Furthermore, Karim et al. (17) found that phosphorylation influenced the proportion of a structurally dynamic form of PLB. To test whether phosphorylation stabilizes a compact pentamer conformation, we obtained distance constraints by measuring fluorescence resonance ...

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

“…This parameter is a measure of the intrinsic FRET of the regulatory complex and is sensitive to conformational changes that alter the distance between the chromophores at the centers of the fluorescent protein tags. With this assay, we have previously quantified small (4 Å) changes in probe separation distance from structure changes induced by phosphomimetic mutations (5,26). However, the present data suggest similar structures for the E1/E2 regulatory complexes.…”

“…4, E and F, were pooled from a set of 470 cells. The data suggest that the observed change in apparent K D 2 (PLB-SERCA) was not an indirect effect of a change in K D 1 (PLB-PLB) (5,6,16,18,26,32) or nonspecific effects on the fluorescent proteins or the membrane structure.…”

“…The hypothesis that PLB inhibition of SERCA can be relieved without dissociation of the regulatory complex is in harmony with studies that have shown that PLB remains bound to SERCA after being phosphorylated (41,42). We have previously compared the effect of phosphomimetic mutations of PLB and phospholemman (PLM) on the respective regulatory complexes with SERCA or the Na ϩ /K ϩ -ATPase (NKA) (5,26). Both of these regulatory complexes persist after phosphomimetic mutations, although with reduced affinity and a small change in the quaternary structure.…”

“…population of cells, as described previously (5,16,17,18,25,26). Observed FRET was compared cell-by-cell to YFP fluorescence, which was taken as an index of protein concentration (5, 16 -18, 26 …”

“…SERCA activity is regulated by phospholamban (PLB), a helical transmembrane peptide that reduces the apparent affinity of the pump for Ca 2ϩ . Inhibition of SERCA by PLB is partially relieved through phosphorylation of PLB by protein kinase A and Ca 2ϩ /calmodulindependent protein kinase (4), which alters the structure of the PLB-SERCA regulatory complex (5) and increases PLB oligomerization into non-inhibitory pentamers (5,6). It is widely recognized that PLB inhibition of SERCA is also relieved by elevated Ca 2ϩ , but the mechanism of this functional effect is unclear.…”