Phospholamban Oligomerization, Quaternary Structure, and Sarco(endo)plasmic Reticulum Calcium ATPase Binding Measured by Fluorescence Resonance Energy Transfer in Living Cells

Abstract

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 ؎ 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 ؎ 0.5 Å , according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K D ) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.The 52-amino acid protein phospholamban (PLB) 2 is an important regulator of cardiac calcium handling (1). PLB binds avidly (2) but reversibly (3) to the sarco(endo)plasmic reticulum calcium ATPase (SERCA), reducing this calcium pump's affinity for calcium (4). PLB also oligomerizes into pentamers through leucine zipper interactions in its transmembrane domain (5, 6). NMR studies indicate that PLB tertiary structure consists of an N-terminal (cytosolic) ␣-helix (domain IA) connected by a flexible loop (domain IB) to a second ␣-helix (domain II) that is anchored in the membrane of the sarcoplasmic reticulum (7). Several possible configurations of the cytoplasmic domains of pentameric PLB have been described. Some of these portray the domain IA ␣-helix as being nearly normal to the surface of the membrane (8, 9), whereas others indicate axial declination of domain IA (7, 10 -14), permitting contact with the surface of the membrane (15, 16). These previous studies were performed using in vitro preparations of PLB in defined lipids or detergent. To investigate the quaternary structure of PLB in the membranes of living cells and distinguish between these structural mo...

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

“…Ser-16 and Thr-17 (the 2 phosphorylatable sites) are exposed to the bulk solvent, prone to interact with their respective kinases (i.e., protein kinase A and CamKII kinase, respectively). The amphipathic interactions of domain Ia with the lipid bilayer are also present in the oligomeric state of PLN, as measured by solution NMR in detergent micelles (14,50), solid-state NMR in lipid bilayers (13,52), EPR saturation transfer experiments in lipid bilayers and detergent micelles (8,12,13), and f luorescence resonance energy transfer in intact cells (46) and detergent reconstituted systems (53).…”

“…In fact, PLN does not possess a compact fold, but instead, has its 3D architecture dictated by interactions with the lipid bilayer. Several in vivo and in vitro studies have shown that PLN adopts a remarkably similar structure both in micelles and lipid bilayers (8,12,15,18,19,25,46,47), justifying the combination of restraints. In the event that restraints obtained in lipid bilayers and detergent micelles were incompatible, it is recommended that those from lipids be weighted more heavily, due to potential limitations of micelles.…”

Phospholamban Binds with Differential Affinity to Calcium Pump Conformers

“…FRET efficiency was calculated according to To estimate the contribution of FRET between non-interacting proteins (nonspecific FRET), we have previously used non-fluorescent PLB to compete for binding. This abolished specific FRET, leaving 4% residual nonspecific FRET (18). In another study, we used a fluorescently labeled PLB that could not participate in FRET as a competitor.…”

“…Cell ScoringWide-field fluorescence microscopy was performed as described previously (18). For each sample, automated acquisition of a field of 48 images was performed using a motorized stage (Prior, Rockland, MA) controlled by the MetaMorph software.…”

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

“…PLB exists as a population of homopentamers and monomers in the SR membrane, the monomer being the active form responsible for Ca 2ϩ -ATPase inhibition (3,4). Several groups have shown that there is a dynamic equilibrium between PLB pentamers, PLB monomers, and PLB-SERCA heterodimers (5)(6)(7)(8)(9). Recent studies with chemical cross-linking have suggested a simple mechanism of PLB inhibition, in which the PLB monomer competes with Ca 2ϩ for binding to SERCA2a by stabilizing a single conformational state of the enzyme ( Fig.…”