Transmembrane Domain Of Phospholamban Research Articles

Nitroxyl (HNO) targets phospholamban cysteines 41 and 46 to enhance cardiac function.

Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Interaction of SERCA with the Transmembrane Domain of Phospholamban Measured by FRET in Live Cells

Phospholamban (PLB) is the key regulator of the SERCA calcium pump. Previous quantitative fluorescence resonance energy transfer (FRET) measurements between Cerulean-SERCA and YFP-PLB have shown that calcium and thapsigargin reduce the affinity of the PLB-SERCA binding interaction. Though a structure change in the regulatory complex would account for this reduced affinity, we detected no change in FRET distance. We hypothesized that in the presence of calcium the transmembrane domain of PLB moves to a secondary site on SERCA without altering the positions of the PLB cytosolic domain or fluorescent protein fusion tag. To test this hypothesis, we engineered a truncated PLB construct (tmPLB) composed of a fluorescent protein fused to the transmembrane domain of PLB (residues 28-52). We observed FRET from Cerulean-SERCA to YFP-tmPLB, but with a significantly longer FRET distance compared to full-length PLB. SERCA-tmPLB FRET was not significantly changed by saturating concentrations of calcium or thapsigargin. Notably, we did not detect inhibition of SERCA by YFP-tmPLB using an in-cell calcium uptake assay, even though other groups have previously shown that the PLB transmembrane domain can inhibit SERCA activity. In addition, oligomerization of the tmPLB was detected by intrapentameric FRET. Compared to full-length PLB, the oligomerization affinity of tmPLB was normal, but we measured a shorter FRET distance as a result of deletion of the cytoplasmic domain. The results demonstrate that tmPLB retains some normal protein-protein interactions despite its apparently anomalous interaction with SERCA. Taken together, the results suggest a non-inhibitory binding interaction between YFP-tmPLB and SERCA, perhaps as a result of steric hindrance by the YFP fusion tag. It is also possible that the truncated PLB binds selectively to a non-inhibitory site on SERCA. Experiments are underway to address these unresolved questions.

Characterizing the Phospholamban-SERCA Complex by Pulsed EPR Spectroscopy

Muscle contraction and relaxation are controlled through the release and reuptake of Ca2+ stored in the sarcoplasmic reticulum (SR). Relaxation is mediated by the SR Ca2+ ATPase (SERCA), a pump that drives Ca2+ against its concentration gradient while hydrolyzing ATP. Cardiac SERCA is regulated by phospholamban (PLB), a small membrane protein that inhibits the pump except when phosphorylated at Ser16. PLB phosphorylation restores SERCA activity without dissociating the two proteins, instead inducing a structural change within the PLB-SERCA complex. Although a number of studies have investigated the interaction of these proteins, the relationship between phosphorylation, structure, and activity remains unresolved. We have used dipolar electron-electron resonance (DEER) spectroscopy, a technique capable of measuring distances from 2-7nm, to characterize large conformational changes within PLB upon phosphorylation and SERCA binding. Our results show that the transmembrane and cytoplasmic helices of PLB draw closer upon SERCA binding, with subsequent phosphorylation compacting the structure still further. However, relative distances between the cytoplasmic domains of PLB and SERCA remain largely constant before and after phosphorylation, suggesting that the observed structural change occurs in the transmembrane domain of PLB. Ultimately, our goal is to make exhaustive distance measurements within the SERCA-PLB complex in order to understand the structural basis of phosphorylation-mediated inhibition relief.

Nitroxyl and “Forbidden Disulfides”: Phospholamban Cysteines are Targeted to Enhance SERCA2a Activity

Our enjoyable collaboration with Dr. Jeffrey P. Froehlich focused on the role of phospholamban (PLN) in HNO-induced enhancement of SR Ca2+-ATPase (SERCA2a) activity. We hypothesized that given HNO thiophilic nature it modifies cysteine residues in PLN transmembrane domain, altering its interaction with SERCA2a, thus enhancing pump activity. When HNO, donated by Angeli's salt (AS), was administered to control isolated myocytes, it enhanced both sarcomere shortening and Ca2+ transient. However, when AS/HNO was applied to PLN-/- ventriculocytes, HNO inotropy was reduced by ≅ 50% (the remaining likely stemming from enhanced myofilament sensitivity to Ca2+). PLN centrality to HNO cardiotropic action was confirmed incubating SR vesicles from WT and PLN-/- mice with AS/HNO to measure ATP-dependent Ca2+ uptake by stopped-flow mixing. In WT, HNO increased Ca2+ uptake rate, but it failed to do so in PLN-/- vesicles. The role of cysteines in PLN emerged from studies using ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46→Ala mutant) where we assessed SERCA2a dephosphorylation, a measure of E2P hydrolysis, i.e. a rate-limiting step of SERCA2a activity. AS/HNO augmented SERCA2a dephosphorylation in ER microsomes co-expressing SERCA2a and WT PLN, but this stimulation was absent in microsomes expressing SERCA2a and Cys 36-41-46→Ala mutant PLN. Thus, Jeff's elegant, creative and passionate approach helped us to show that PLN is essential for HNO-induced faster Ca2+ uptake by SERCA2a, suggesting that HNO action occurs, at least partly, via modifications of critical cysteines in PLN transmembrane domain. In Jeff's view, “forbidden” disulfide bonds in PLN are involved, and one of his legacies for us is unearthing the cysteine pairs that are involved in HNO-induced formation of an intramolecular bond that could distort the conformation of PLN, thus perturbing its interaction with SERCA2a and relieving the inhibition.

Effects of Phospholamban Transmembrane Mutants on the Calcium Affinity, Maximal Activity, and Cooperativity of the Sarcoplasmic Reticulum Calcium Pump

Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to interactions between their respective transmembrane domains. In spite of numerous mutagenesis and kinetic studies, we still do not have a clear mechanistic picture of how PLB influences the calcium transport cycle of SERCA. Herein, we have created alanine mutants for each residue in the transmembrane domain of PLB, we have co-reconstituted these mutants with SERCA into proteoliposomes, and we have performed kinetic simulations of the calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a variable effect on the calcium affinity, maximal activity, and cooperativity of SERCA, such that a range of values was observed. Kinetic simulations using a well-established reaction scheme for SERCA then allowed us to correlate the effects on SERCA activity with changes in the reaction scheme rate constants. Only three steps in the reaction scheme were affected by the presence of PLB, namely, binding of the first calcium ion, a subsequent conformational change in SERCA, and binding of the second calcium ion. The ability of wild-type and mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated with a decreased rate of binding of the second calcium ion. In addition, the ability of wild-type and mutant forms of PLB to alter the maximal activity of SERCA correlated with a change in the forward rate constant for the slow conformational change in SERCA following binding of the first calcium ion.

Peptide Inhibitors Use Two Related Mechanisms To Alter the Apparent Calcium Affinity of the Sarcoplasmic Reticulum Calcium Pump

The primary sequence of phospholamban (PLB) has provided a template for the rational design of peptide inhibitors of the sarcoplasmic reticulum calcium ATPase (SERCA). In the transmembrane domain of PLB, there are few polar residues and only one is essential (Asn (34)). Using synthetic peptides, we have previously investigated the role of Asn (34) in the context of simple hydrophobic transmembrane peptides. Herein we propose that the role of Asn in SERCA inhibition is position-sensitive and dependent upon the distribution of hydrophobic residues. To test this hypothesis, we synthesized a series of transmembrane peptides based on a 24 amino acid polyalanine sequence having either an alternating Leu-Ala sequence (Leu 12) or Leu residues at the native positions found in PLB (Leu 9). Asn-containing Leu 9 and Leu 12 peptides were synthesized with a single Asn residue located either one amino acid (N+/-1) or one turn of the helix (N+/-4) in either direction from its native position. Co-reconstitution of these peptides with SERCA into proteoliposomes revealed effects on the apparent calcium affinity and cooperativity of SERCA that correlated with the positions of the Asn and Leu residues. The most inhibitory peptides increased the cooperativity of SERCA as indicated by the Hill coefficients, suggesting that calcium-dependent reversibility is an inherent part of the inhibitory mechanism. Kinetic simulations combined with molecular modeling of the interaction between the peptides and SERCA reveal two related mechanisms of inhibition. Peptides that resemble PLB use the same inhibitory mechanism, whereas peptides that are more divergent from PLB alter an additional step in the calcium transport cycle.

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

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 +/- 0.5A(,) 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.

Mechanism of Reversal of Phospholamban Inhibition of the Cardiac Ca2+-ATPase by Protein Kinase A and by Anti-phospholamban Monoclonal Antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.

Two-Dimensional Solid-State NMR Reveals Two Topologies of Sarcolipin in Oriented Lipid Bilayers

Sarcolipin (SLN), a 31 amino acid integral membrane protein, regulates SERCA1a and SERCA2a, two isoforms of the sarco(endo)plasmic Ca-ATPase, by lowering their apparent Ca(2+) affinity and thereby enabling muscle relaxation. SLN is expressed in both fast-twitch and slow-twitch muscle fibers with significant expression levels also found in the cardiac muscle. SLN shares approximately 30% identity with the transmembrane domain of phospholamban (PLN), and recent solution NMR studies carried out in detergent micelles indicate that the two polypeptides bind to SERCA in a similar manner. Previous 1D solid-state NMR experiments on selectively (15)N-labeled sites showed that SLN crosses the lipid bilayer with an orientation nearly parallel to the bilayer normal. With a view toward the characterization of SLN structure and its interactions with both lipids and SERCA, herein we report our initial structural and topological assignments of SLN in mechanically oriented DOPC/DOPE lipid bilayers as mapped by 2D (15)N PISEMA experiments. The PISEMA spectra obtained on uniformly (15)N-labeled protein as well as (15)N-Leu, (15)N-Ile and (15)N-Val map the secondary structure of SLN and, simultaneously, reveal that SLN exists in two distinct topologies. Both the major and the minor populations assume an orientation with the helix axis tilted by approximately 23 degrees with respect to the lipid bilayer normal, but vary in the rotation angle about the helix axis by approximately 5 degrees . The existence of the multiple populations in model membranes may be a significant requirement for SLN interaction with SERCA.

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.

Cross-linking of C-terminal Residues of Phospholamban to the Ca2+ Pump of Cardiac Sarcoplasmic Reticulum to Probe Spatial and Functional Interactions within the Transmembrane Domain

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.

Investigating Structural Changes in the Lipid Bilayer upon Insertion of the Transmembrane Domain of the Membrane-Bound Protein Phospholamban Utilizing 31P and 2H Solid-State NMR Spectroscopy

Phospholamban (PLB) is a 52-amino acid integral membrane protein that regulates the flow of Ca 2+ ions in cardiac muscle cells. In the present study, the transmembrane domain of PLB (24–52) was incorporated into phospholipid bilayers prepared from 1-palmitoyl-2-oleoyl- sn-glycero-phosphocholine (POPC). Solid-state 31P and 2H NMR experiments were carried out to study the behavior of POPC bilayers in the presence of the hydrophobic peptide PLB at temperatures ranging from 30°C to 60°C. The PLB peptide concentration varied from 0 mol % to 6 mol % with respect to POPC. Solid-state 31P NMR spectroscopy is a valuable technique to study the different phases formed by phospholipid membranes. 31P NMR results suggest that the transmembrane protein phospholamban is incorporated successfully into the bilayer and the effects are observed in the lipid lamellar phase. Simulations of the 31P NMR spectra were carried out to reveal the formation of different vesicle sizes upon PLB insertion. The bilayer vesicles fragmented into smaller sizes by increasing the concentration of PLB with respect to POPC. Finally, molecular order parameters ( S CD) were calculated by performing 2H solid-state NMR studies on deuterated POPC ( sn-1 chain) phospholipid bilayers when the PLB peptide was inserted into the membrane.

Sarcolipin regulates sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) by binding to transmembrane helices alone or in association with phospholamban.

Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E(2) conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.

Helical structure of phospholamban in membrane bilayers

The regulation of calcium levels across the membrane of the sarcoplasmic reticulum involves the complex interplay of several membrane proteins. Phospholamban is a 52 residue integral membrane protein that is involved in reversibly inhibiting the Ca2+ pump and regulating the flow of Ca ions across the sarcoplasmic reticulum membrane during muscle contraction and relaxation. The structure of phospholamban is central to its regulatory role. Using homonuclear rotational resonance NMR methods, we show that the internuclear distances between [1-13C]Leu7 and [3-13C]Ala11 in the cytoplasmic region, between [1-13C]Pro21 and [3-13C]Ala24 in the juxtamembrane region and between [1-13C]Leu42 and [3-13C]Cys46 in the transmembrane domain of phospholamban are consistent with α-helical secondary structure. Additional heteronuclear rotational-echo double-resonance NMR measurements confirm that the secondary structure is helical in the region of Pro21 and that there are no large conformational changes upon phosphorylation. These results support the model of the phospholamban pentamer as a bundle of five long α-helices. The long extended helices provide a mechanism by which the cytoplasmic region of phospholamban interacts with residues in the cytoplasmic domain of the Ca2+ pump.

Role of Cysteine Residues in Structural Stability and Function of a Transmembrane Helix Bundle

To study the structural and functional roles of the cysteine residues at positions 36, 41, and 46 in the transmembrane domain of phospholamban (PLB), we have used Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase peptide synthesis to prepare alpha-amino-n-butyric acid (Abu)-PLB, the analogue in which all three cysteine residues are replaced by Abu. Whereas previous studies have shown that replacement of the three Cys residues by Ala (producing Ala-PLB) greatly destabilizes the pentameric structure, we hypothesized that replacement of Cys with Abu, which is isosteric to Cys, might preserve the pentameric stability. Therefore, we compared the oligomeric structure (from SDS-polyacrylamide gel electrophoresis) and function (inhibition of the Ca-ATPase in reconstituted membranes) of Abu-PLB with those of synthetic wild-type PLB and Ala-PLB. Molecular modeling provides structural and energetic insight into the different oligomeric stabilities of these molecules. We conclude that 1) the Cys residues of PLB are not necessary for pentamer formation or inhibitory function; 2) the steric properties of cysteine residues in the PLB transmembrane domain contribute substantially to pentameric stability, whereas the polar or chemical properties of the sulfhydryl group play only a minor role; 3) the functional potency of these PLB variants does not correlate with oligomeric stability; and 4) acetylation of the N-terminal methionine has neither a functional nor a structural effect in full-length PLB.

A solid-state NMR study of the phospholamban transmembrane domain: local structure and interactions with Ca 2+-ATPase

The structure and dynamics of a double 13C-labelled 24-residue synthetic peptide ([ 13C 2]CAPLB 29–52), corresponding to the membrane-spanning sequence of phospholamban (PLB), were examined using 13C cross-polarisation magic-angle spinning (CP-MAS) NMR spectroscopy. CP-MAS spectra of [ 13C 2]CAPLB 29–52 reconstituted into unsaturated lipid membranes indicated that the peptide was mobile at temperatures down to −50°C. The NMR spectra showed that peptide motion became constrained in the presence of the SERCA1 isoform of Ca 2+-ATPase, and chemical cross-linking experiments indicated that [ 13C 2]CAPLB 29–52 and Ca 2+-ATPase came into close contact with one another. These results together suggested that the peptide and the 110-kDa calcium pump were interacting in the membrane. Rotational resonance CP-MAS 13C– 13C distance measurements on [ 13C 2]CAPLB 29–52 reconstituted into lipid bilayers confirmed that the sequence spanning Phe-32 and Ala-36 was α-helical, and that this structure was not disrupted by interaction with Ca 2+-ATPase. These results support the finding that the transmembrane domain of PLB is partially responsible for regulation of Ca 2+ transport through interactions with cardiac muscle Ca 2+-ATPase in the lipid bilayer, and also demonstrate the feasibility of performing structural measurements on PLB peptides when bound to their physiological target.

Synthetic null-cysteine phospholamban analogue and the corresponding transmembrane domain inhibit the Ca-ATPase.

Chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) have been used to analyze the structure and function of phospholamban (PLB), a 52-residue integral membrane protein that regulates the calcium pump (Ca-ATPase) in cardiac sarcoplasmic reticulum (SR). PLB exists in equilibrium between monomeric and pentameric forms, as observed by SDS-PAGE, EPR, and fluorescence. It has been proposed that inhibition of the pump is due primarily to the monomeric form, with both pentameric stability and inhibition dependent primarily on the transmembrane (TM) domain. To test these hypotheses, we have studied the physical and functional properties of a synthetic null-cysteine PLB analogue that is entirely monomeric on SDS-PAGE, and compared it with the synthetic null-cysteine TM domain (residues 26-52). The TM domain was found to be primarily oligomeric on SDS-PAGE, and boundary lipid spin label analysis in lipid bilayers verified that the isolated TM domain is more oligomeric than the full-length parent molecule. These results indicate that the stability of the PLB pentamer is due primarily to attractive interactions between hydrophobic TM domains, overcoming the repulsive electrostatic interactions between the cationic cytoplasmic domains (residues 1-25). When reconstituted into liposomes containing the Ca-ATPase, the null-cysteine TM domain had the same inhibitory function as that of the full-length parent molecule. We conclude that the TM domain of PLB is sufficient for inhibitory function, the oligomeric stability of PLB does not determine its inhibitory activity, and the three Cys residues in the TM domain are not required for inhibitory function.

Toward a high-resolution structure of phospholamban: design of soluble transmembrane domain mutants.

Determination of a high-resolution structure of the phospholamban (PLB) transmembrane domain by X-ray crystallography or NMR is handicapped by the hydrophobic nature of the peptide. Interestingly, the crystal structure of the five-stranded parallel coiled-coil oligomerization domain from cartilage oligomeric matrix protein (COMPcc) shows marked similarities to a model proposed for the pentameric transmembrane domain of PLB. Contrary to the putative coiled-coil domain of PLB, COMPcc contains mostly hydrophilic amino acids on the surface, resulting in a soluble molecule. Here, we report the design of soluble PLB transmembrane domain variants by combining the surface residues of COMPcc and the hydrophobic interior of the transmembrane domain of PLB. The soluble PLB variants formed pentameric structures as revealed by analytical ultracentrifugation. After redox shuffling, they showed unspecific disulfide bridge patterns similar to that of the chemically synthesized wild-type PLB transmembrane domain. These results suggest a structural homology between the soluble PLB mutants and the wild-type PLB transmembrane domain. Together with the data reported in the literature, they furthermore indicate that residues Leu37, Ile40, Leu44, and Ile47 of the PLB sequence specify pentamer formation. In contrast, a designed recombinant COMPcc mutant, COMP-ARCC, which was engineered to contain the two PLB cysteines that potentially could form an interchain disulfide bridge, formed a specific disulfide bond pattern. This finding indicates structural differences between the transmembrane domain of PLB and COMPcc. The soluble PLB variants may be used to determine a high-resolution structure of the PLB pentamer by X-ray crystallography.

Sites of Regulatory Interaction between Calcium ATPases and Phospholambana

Phospholamban (PLN) is a 52-amino acid, integral membrane protein that interacts with and reversibly inhibits the activity of the cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a). We have used site-directed mutagenesis to analyze the sites of interaction between PLN and SERCA2a. First, we used chimera formation between SERCA2a and SERCA3 (which is weakly inhibited by PLN) to determine the interacting residues in cytoplasmic sequences of SERCA2 and PLN. Then, we expressed SERCA2a with the transmembrane sequence of PLN and demonstrated that the sites of inhibitory interaction are located in transmembrane sequences of the two proteins. We proposed that a four-base circuit involving noninhibitory cytoplasmic and inhibitory transmembrane sites in PLN and SERCA2a best describes the interaction. Recently, we have used alanine-scanning mutagenesis to show an asymmetric distribution of function in the transmembrane domain of PLN--one helical face interacts with PLN molecules in a pentamer, and the other interacts with SERCA2a. Gain of function by mutation of PLN-interacting residues indicates that the inhibitory species of PLN is a monomer. Thus regulatory steps include PLN dissociation, PLN/SERCA2a inhibitory association, and PLN/SERCA2a dissociation induced by phosphorylation of PLN (in the noninhibitory cytoplasmic domain) or by binding of Ca2+ by SERCA2a (in the inhibitory transmembrane domain).

Direct spectroscopic detection of molecular dynamics and interactions of the calcium pump and phospholamban.

In order to test molecular models of cardiac calcium transport regulation, we have used spectroscopy to probe the structures, dynamics, and interactions of the Ca pump (Ca-ATPase) and phospholamban (PLB) in cardiac sarcoplasmic reticulum (SR) and in reconstituted membranes. Electron paramagnetic resonance (EPR) and phosphorescence of probes bound to the Ca pump show that the activity of the pump is quite sensitive to its oligomeric interactions. In cardiac SR, PLB aggregates and inhibits the pump, and both effects are reversed by PLB phosphorylation. Previous analyses of PLB's oligomeric state were only in detergent solutions, so we used EPR and fluorescence to determine the oligomeric structure of PLB in its native state in lipid bilayers. Wild-type PLB is primarily oligomeric in the membrane, while the mutant L37A-PLB is monomeric. For both proteins, phosphorylation shifts the dynamic monomer-oligomer equilibrium toward oligomers, and induces a similar structural change, as indicated by tyrosine fluorescence; yet L37A-PLB is more effective than wild-type PLB in inhibiting and aggregating the pump. Fluorescence energy transfer shows that the Ca pump increases the fraction of monomeric PLB, indicating that the pump preferentially binds monomeric PLB. These results support a reciprocal aggregation model for Ca pump regulation, in which the Ca pump is aggregated and inhibited by association with PLB monomers, and phosphorylation of PLB reverses these effects while decreasing the concentration of PLB monomers. To investigate the structure of the PLB pentamer in more detail, we measured the reactivities of cysteine residues in the transmembrane domain of PLB, and recorded EPR spectra of spin labels attached to these sites. These results support an atomic structural model, based on molecular dynamics simulations and mutagenesis studies, in which the PLB pentamer is stabilized by a leucine-isoleucine zipper within the transmembrane domain.