Phospholamban Oligomer Research Articles

The Phospholamban Pentamer Functionally Interacts with the Sarcoplasmic Reticulum Calcium Pump SERCA

The interaction of phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric phospholamban and storage of phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that phospholamban remains associated with SERCA and that the phospholamban pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of phospholamban also interacts with M3 of SERCA, though the phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of phospholamban interacts with M3 of SERCA, and that it plays distinct structural and functional roles in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of phospholamban at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for altering the turnover rate of SERCA.

Two dimensional crystallization of calcium transport regulatory complexes

Phospholamban and sarcolipin physically interact with the sarcoplasmic reticulum calcium pump (SERCA) and differentially regulate contractility in smooth, skeletal, and cardiac muscle. While mutagenesis and cross-linking studies have provided insight into the mechanism of interaction, we lack a molecular understanding of these regulatory complexes. We have compared two crystal forms of SERCA in the absence and presence of phospholamban by electron cryo-microscopy. Our previous studies with phospholamban utilized both small helical crystals [1] and large two-dimensional crystals [2], where the fundamental units of both crystal forms were found to be anti-parallel dimer ribbons of SERCA molecules. The SERCA dimer ribbons have been known for decades as a rigid assembly of calcium-free SERCA molecules induced by the addition of decavanadate. While the lattice formed by the SERCA dimer ribbons is different in the helical (p2) and two-dimensional crystals (p22121), we now show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the large two-dimensional crystals. Both wild-type and a gain-of-function mutant (Asn4-to-Ala) mutant of sarcolipin were utilized. Projection maps were determined for SERCA in the presence of sarcolipin to a resolution of 8.5 Å and were most consistent with a pentameric state for sarcolipin. While both phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a sarcolipin monomer.

Phospholamban and Sarcolipin Pentamers Naturally Associate with the Sarcoplasmic Reticulum Calcium Pump

Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both phospholamban and sarcolipin naturally associate with SERCA.

Phosphorylation and Mutation Induce an Order-To-Disorder Transition in the Cytoplasmic Domain of Phospholamban

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with phospholamban. In previous studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was disordered. The functional state of phospholamban correlated with an order-to-disorder transition of phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, the residues studied (Ser16, Lys27 and Asn34) may implicate domain Ib of phospholamban in the order-to-disorder transition. In summary, the two-dimensional co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association. Phosphorylation or mutation of phospholamban alters the SERCA-pentamer interaction in a manner normally associated with inhibition by the monomer.

Structural Dynamics of SERCA and Phospholamban by TR-FRET and TIRF

We used fluorescence to investigate the structure and dynamics of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Time-resolved fluorescence energy transfer (TR-FRET) was used to study the oligomeric interaction between PLB and SERCA. Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. We used polarized TIRF to study the structure of PLB in the presence and absence of SERCA in a supported lipid bilayer. Polarized TIRF data shows that the cytoplasmic helix of PLB, labeled with bifunctional rhodamine, is close to be parallel to the membrane surface. SERCA binding lifts the cytoplasmic helix away from the membrane surface. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.

Phosphorylation and Mutation of Phospholamban Alter Physical Interactions With the Sarcoplasmic Reticulum Calcium Pump

Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.

A Functional, Pentameric Form of Phospholamban Is Required For Two-Dimensional Crystallization With the Sarcoplasmic Reticulum Calcium Pump

A Functional, Pentameric Form Of Phospholamban Is Required For Two-Dimensional Crystallization With The Sarcoplasmic Reticulum Calcium PumpPhospholamban physically interacts with the sarcoplasmic reticulum calcium pump (also known as SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of large two-dimensional crystals of SERCA in complex with phospholamban. In our original work, phospholamban oligomers were found interspersed between dimer arrays of SERCA and a three-dimensional model was constructed to show potential interactions between the two proteins. In the present study, we have examined the effects of phospholamban phosphorylation and mutation on the formation of two-dimensional co-crystals with SERCA. Phospholamban phosphorylation at Ser16 and a well-characterized loss-of-function mutation (Asn34-to-Ala) significantly reduced crystal formation. More importantly, projection maps calculated from these crystals revealed that the densities attributable to phospholamban become disordered, suggesting a reduced interaction with SERCA. In contrast, a pentameric gain-of-function mutant (Lys27-to-Ala) significantly enhanced crystal formation. These latter crystals were used to calculate an improved projection map from frozen-hydrated crystals to a resolution of 8 A. We conclude that the oligomeric state of phospholamban in the crystals is a pentamer, and that phosphorylation and mutation of phospholamban alter physical interactions in the crystals in a manner that is consistent with a functional association with SERCA. Combined, the data suggest that the pentameric state of phospholamban is not simply an inactive storage form.

Oligomeric Interactions in Cardiac Calcium Regulation

We used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the oligomeric state of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.

Rational Design of Peptide Inhibitors of the Sarcoplasmic Reticulum Calcium Pump

The sequence of phospholamban (PLB) is practically invariant among mammalian species. The hydrophobic transmembrane domain has 10 leucine and 8 isoleucine residues. Two roles have been proposed for the leucines; one subset stabilizes PLB oligomers, while a second subset physically interacts with SERCA. On the basis of the sequence of the PLB transmembrane domain, we chemically synthesized a series of peptides and tested their ability to regulate SERCA in reconstituted membranes. In all, eight peptides were studied: a peptide corresponding to the null-cysteine transmembrane domain of PLB (TM-Ala-PLB), two polyleucine peptides (Leu18 and Leu24), polyalanine peptides containing 4, 7, and 12 leucine residues (Leu4, Leu7, and Leu12, respectively), and a polyalanine peptide containing the 9 leucine residues present in the transmembrane domain of PLB with and without the essential Asn34 residue (Asn1Leu9 and Leu9, respectively). With the exception of Leu18, co-reconstitution of the peptides revealed effects on the apparent calcium affinity of SERCA. The TM-Ala-PLB peptide possessed approximately 70% of the inhibitory function of wild-type PLB. The remaining peptides exhibited significant inhibitory activity decreasing in the following order: Leu12, Leu9, Leu24, Leu7, and Leu4. Replacing Asn34 of PLB in the Leu9 peptide resulted in superinhibition of SERCA. On the basis of these observations, we conclude that a partial requirement for SERCA inhibition is met by a simple hydrophobic surface on a transmembrane alpha-helix. In addition, the superinhibition observed for the Asn34-containing peptide suggests that the model peptides mimic the inhibitory properties of PLB. A model is presented in which surface complementarity around key amino acid positions is enhanced in the interaction with SERCA.

Depolymerization of phospholamban in the presence of calcium pump: a fluorescence energy transfer study.

Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by beta-adrenergic stimulation. The mobility of PLB on SDS-PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9-11 subunits, with approximately 10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 +/- 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30-40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.

Mutation and phosphorylation change the oligomeric structure of phospholamban in lipid bilayers.

Phospholamban (PLB), a 52-residue protein integral to the cardiac sarcoplasmic reticulum, is a key regulator of the Ca pump. PLB has been shown to form pentamers in the denaturing detergent sodium dodecyl sulfate (SDS), but its oligomeric state in the natural environment of the lipid membrane remains unknown. In order to address this issue, we performed electron paramagnetic resonance (EPR) experiments on two types of lipid-reconstituted, recombinant PLB: wild type (WT PLB) and a mutant substituted with alanine at leucine 37 (L37A PLB), whose propensity to oligomerize in SDS is greatly diminished. The lipid used in reconstitution was dioleoylphosphatidylcholine (DOPC) doped with a phospholipid spin-label that detects protein contact. EPR spectroscopy was used to determine the fraction of the total lipid molecules in contact with PLB. Our results show that, in phospholipid bilayers, WT PLB is oligomeric (effective oligomeric size of 3.52 +/- 0.71), while L37A PLB is monomeric (effective oligomeric size of 1.15 +/- 0.15). Thus, the oligomeric states of these proteins in the lipid membrane are remarkably similar to those in SDS solution. In particular, the point mutation in L37A PLB greatly destabilizes the PLB oligomer. Phosphorylation of PLB by protein kinase A, which has been shown to relieve inhibition of the cardiac Ca pump, changes the lipid-PLB interactions, decreasing the number of lipids restricted by contact with protein. The results are consistent with a phosphorylation-dependent increase of the effective oligomer size of WT PLB from 3.52 to 5.34 and of L37A PLB from 1.15 to 1.91. These phosphorylation effects were abolished in a medium with a high ionic strength. We conclude that the oligomeric states of PLB in lipid membranes are in a dynamic equilibrium that is perturbed by phosphorylation due to reduced electrostatic repulsion among PLB protomers.

Molecular Weight Determination of Phospholamban Oligomer in the Presence of Sodium Dodecyl Sulfate: Application of Low-Angle Laser Light Scattering Photometry1

The number of polypeptides constituting the oligomeric structure of canine phospholamban (a putative regulator of Ca(2+)-ATPase of cardiac sarcoplasmic reticulum) stable even in the presence of sodium dodecyl sulfate was estimated through determination of the molecular weight of the oligomer. Owing to the small molecular size, the low UV-absorptivity and the limited availability, the molecular weight determination required very sophisticated application of the following technique, used as the only recourse: low-angle laser light scattering measurement combined with high-performance gel chromatography. The molecular weight of phospholamban oligomer was found to be 30,400 and the number of subunits was concluded to be five after correction for the dependence of the apparent molecular weights on the protein concentration.

Immunological detection of phospholamban phosphorylation states facilitates the description of the mechanism of phosphorylation and dephosphorylation

Six electrophoretically distinct species of oligomeric phospholamban were identified immunologically following phosphorylation of sarcoplasmic reticulum vesicles by cAMP-dependent protein kinase. The phosphate content of each was determined, confirming that the discrete sequential retardation of phospholamban oligomers was the result of ascending mole ratios of phosphate (P0-P5) per oligomer. These data afford support to the pentameric arrangement of oligomeric phospholamban and offer a means of determining phosphorylation stoichiometry independent of the absolute phospholamban concentration. Detection of the relative concentration of individual species during phosphorylation facilitated the description of a random mechanism of phosphorylation by cAMP-dependent protein kinase. By contrast, dephosphorylation of cAMP-dependent protein kinase phosphorylated phospholamban was shown to exhibit strong positive cooperativity in its reaction mechanism.

Concerted phosphorylation of the 26-kilodalton phospholamban oligomer and of the low molecular weight phospholamban subunits.

Phospholamban (PLB) from cardiac sarcoplasmic reticulum (SR) was phosphorylated under various conditions by the adenosine cyclic 3',5'-phosphate (cAMP)-dependent and/or the calmodulin-dependent protein kinase. The small shifts in apparent molecular weight resulting from the incorporation of Pi groups in the PLB complexes were analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In parallel experiments, PLB was dissociated into its subunits and analyzed by using a newly developed isoelectric focusing system. The pI values of the PLB subunits phosphorylated by the cAMP- or calmodulin-dependent kinase were 6.2 and 6.4, respectively. Double phosphorylation of the same subunit resulted in an acidic shift of the pI to 5.2. The combined analysis of the behavior of the PLB complex and of its subunits has greatly simplified the interpretation of the complex phosphorylation pattern and has led to the following conclusions: The PLB complex is composed of five probably identical subunits, each of them containing a distinct phosphorylation site for the calmodulin- and the cAMP-dependent kinase. The population of PLB interacting with the endogenous calmodulin-dependent kinase cannot be phosphorylated by the cAMP-dependent kinase unless previously phosphorylated in the presence of calmodulin. It was also observed that after maximal phosphorylation of PLB in the presence of very large amounts of the cAMP-dependent protein kinase, the Ca2+ pumping rate of the cardiac SR ATPase is stimulated up to 5-fold, i.e., a level of a stimulation which exceeds considerably the values so far reported in the literature.