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We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca(2+)-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca(2+)-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca(2+)-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K(+) and 3 mM Mg(2+). Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K(+) ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K(+) in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H(+)771. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca(2+)-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump.

We have used x-ray crystallography to solve the crystal structure of the sarco/endoplasmic reticulum Ca-ATPase (SERCA) in complex with a novel lead compound (CXL017) that targets multidrug-resistant leukemia (Hermanson et al., Mol Pharmacol 2009). The structure, solved at 3.01A resolution, reveals CXL017 bound to SERCA near the ATP binding site and suggests blockage of the ATP channel as the main mechanism of inhibition. This result confirms that CXL017 binds SERCA at a location that is distinct from the binding sites of other inhibitors of SERCA, and it explains the synergistic relationship that was uncovered between CXL017 and classic SERCA inhibitors (Bleeker et al., Mol. Pharmacol 2013). Further, it provides the platform for structure-guided improvements on this new class of potential anti-cancer agent. This work was funded by a grant from NIH to DDT (R01 GM27906) and a grant from NIH to XC (R01 CA163864).

We have used a comprehensive array of techniques to characterize the energy-transduction domain of the sarco/endoplasmic reticulum calcium transport ATPase (SERCA), including x-ray crystallography, solution biochemistry, fluorescence spectroscopy, molecular dynamics simulation, and small-molecule activation. The energy-transduction domain of SERCA mediates long-range allosteric coupling of ATP hydrolysis in the cytosolic headpiece to Ca2+ transport in the transmembrane domain. In heart failure, decreased SERCA activity correlates with disease progression in patients; increasing SERCA activity by drug or gene therapy reverses heart failure in animal models. Here we solved the x-ray crystal structure of SERCA in complex with a novel small-molecule activator, thus identifying a unique binding site on the energy-transduction domain. Conformation-specific proteolytic cleavage and intramolecular glutaraldehyde cross-linking was used to determine activator effect on SERCA headpiece structure. Fluorescence spectroscopy was used to identify kinetic transitions accelerated by the activator. We propose a structural and biochemical mechanism for small-molecule activation of SERCA. Interestingly, the activator site is located vicinal to the binding site of sarcolipin (SLN), a muscle phosphoprotein that regulates SERCA energy expenditure and muscle metabolism to provide resistance against diet-induced obesity and extreme cold through non-shivering thermogenesis. SLN uncoupling of ATP hydrolysis from Ca2+ transport by SERCA was examined using microsecond MD simulation, finding that the cytosolic domain of SLN induces a salt bridge-mediated structural rearrangement of the energy-transduction domain, which allosterically uncouples SERCA by disrupting Ca2+ occlusion at residue E309 in transport site II, and thereby facilitating futile Ca2+ backflux in muscle. Our results demonstrate that the energy-transduction domain is a key control element that provides a structural motif for activation of Ca2+ transport by cardiac SERCA2a in heart failure and for uncoupling ATP hydrolysis by muscle SERCA1a in obesity.

We developed a high-precision high-throughput live-cell assay to screen for small molecules that activate the non-muscle sarco/endo-plasmic reticulum calcium ATPase (SERCA2b) using a “2-color” SERCA2b biosensor and a fluorescence-lifetime plate reader (FLT-PR). The 2-color SERCA2b (h2CS2b) biosensor was derived from a human pancreatic SERCA isoform, in which red fluorescent protein (mRuby2) was fused to the N terminus and green fluorescent protein (Clover) to an interior loop. A HEK cell line expressing the h2CS2b biosensors maintained stable expression level of the biosensors for several tens of passages, ideal for large scale HTS campaigns. This biosensor, upon binding of SERCA2b effectors, translates the minute SERCA structural rearrangements into small changes of intramolecular FRET that can only be reliably detected using fluorescence lifetime (FLT). The h2CS2b biosensor provided dose-dependent FRET changes in response to SERCA inhibitors such as thapsigargin, CPA, and BHQ, and SERCA activators previously discovered in this lab. Two instruments developed by Fluorescence Innovations were crucial for this work: a FLT plate reader, which provides a 30-fold increase of precision compared to conventional intensity-based instruments, and a high-speed fluorescence-spectra plate reader, which was used to filter out artifacts due to compound fluorescence. We screened a commercial chemical library of 50000 compounds and identified 94 compounds that significantly affect 2-color SERCA fluorescence lifetime. Functional assays are underway to evaluate the biochemical properties of these small molecules, and their potential for therapeutic application in diabetes and heart failure. This type of assay is being applied to several other drug targets, with promising results. This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220), and to GDG (STTR,5R41DA037622-02).

We used subnanosecond-resolved fluorescence and all-atom molecular dynamics (MD) simulation to detect structural dynamics of the sarcoplasmic reticulum calcium/proton counter-transport ATPase (SERCA) and its inhibitory interactions with sarcolipin (SLN) and phospholamban (PLB). SLN and PLB individually regulate SERCA through direct interaction; SLN is highly expressed in fast-twitch muscle and atria, while PLB is highly expressed in slow-twitch muscle and ventricles. When SERCA, SLN, and PLB are expressed in the same human cell (vastus lateralis muscle, Takotsubo cardiomyopathy, ventricular mitral regurgitation), the three proteins form a “super-inhibitory” ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximal velocity. Forster resonance energy transfer (FRET) was used to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SLN, PLB, and SERCA1a: five binary interactions were assayed and three assembly parameters were determined (binding affinity, oligomer number, interprobe distance). FRET results indicate that SLN and PLB show high-affinity self-association into homo-oligomers, and that SERCA forms heterodimers with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. To determine inhibition mechanisms of subunit-bound complexes, we used microsecond MD simulation of SERCA1a ± SLN or PLB in a POPC bilayer, with calcium, proton, or magnesium ions bound to transport sites of the SERCA transmembrane domain. MD results indicate that two potassium ions initially bind and pre-organize transport sites for calcium binding (SERCA activation), and that SLN and PLB individually inhibit SERCA by populating a calcium/potassium-free intermediate state with one bound proton at Glu771 (transport site I). We propose that super-inhibition of SERCA in the ternary complex is mediated by SLN/PLB-synergistic inhibition of proton/potassium exchange. Acknowledgments: this work was funded by grants to DDT (NIH-GM27906) and MEF (AHA-12SDG12060656).

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells (Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1.Ca to E1'.Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu(37) is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37 degrees C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity ( approximately 0.75 micromol. nmol Ca-ATPase(-1).min(-1) at 15 microM Ca(2+)), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0 degrees C, using 1 microM MgATP, the K(Ca) values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37 degrees C. Measurements of the time course of phosphoenzyme formation at 0 degrees C, using 1 microM MgATP and 268 nM ionized [Ca(2+)], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca(2+) ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E.Ca to E'.Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca(2+) binding to the E1 intermediate.

We have detected structural dynamics in the nucleotide-binding domain of the Ca-ATPase (SERCA) using fluorescence spectroscopy and molecular modeling. SERCA in sarcoplasmic reticulum (SR) vesicles was selectively labeled with fluorescein isothiocyanate (FITC), an affinity probe that specifically reacts with Lys-515 in the nucleotide-binding site. FITC-labeled SERCA enters the phosphorylated “E1P-like” conformation with low probe fluorescence (∼50% intensity) when SR vesicles are loaded with calcium using acetyl phosphate as the energy source, followed by chelation of extravesicular calcium to halt enzyme cycling. Here we compared FITC-SERCA in the unique low fluorescence state (E1P-like) with conformations that show high probe fluorescence: calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Direct waveform recording (DWR) and time-correlated single photon counting (TCSPC) were used to measure fluorescence emission decay and time-resolved anisotropy. Iodide quenching and detergent solubilization were used to examine FITC accessibility and SERCA oligomerization. Spectroscopy results indicate that FITC-SERCA in the E1P-like state shows increased probe dynamics, decreased probe accessibility, and decreased SERCA oligomerization in comparison to high fluorescence states (E2, E1, EP). Molecular modeling of protein and probe conformation identified Phe-487 in the nucleotide-binding domain as a critical residue involved in FITC binding, contributing to the high specificity of labeling for SERCA. Phe-487 also serves as the key residue that ‘stacks’ with the adenine moiety of ATP bound to unlabeled SERCA. We combine fluorescence data and modeling results to propose a structural mechanism for ADP release following ATP hydrolysis and E1P formation by SERCA. This work was funded by grant from NIH to DDT (R01 GM27906).

We have used fluorescence microscopy to detect interactions of sarcolipin (SLN), phospholamban (PLB), and the SR Ca-ATPase (SERCA1a isoform). SLN and PLB individually regulate SERCA activity through protein-protein interactions controlled by phosphorylation; SLN is highly expressed in fast-twitch muscle and atria, while PLB is highly expressed in slow-twitch muscle and ventricles. When SERCA, SLN, and PLB are expressed in the same human cell (vastus lateralis muscle, Takotsubo cardiomyopathy), the three proteins form a “super-inhibited” ternary complex (SERCA-SLN-PLB), whereby SERCA activity shows 5-fold decreased calcium affinity and 2-fold decreased maximum velocity (MacLennan JBC 2002; Tupling PLOS 2013). Here we used Forster resonance energy transfer (FRET) to quantify the complex equilibria of homo- and hetero-oligomeric interactions between SERCA, SLN, and PLB. The three proteins were tagged with genetically-encoded fluorescent probes (CFP, YFP), and the fluorescent fusion proteins were expressed in Sf21 cells via baculovirus infection. Five protein-protein interactions were assayed (SLN-SLN, SLN-PLB, PLB-PLB, SERCA-SLN, SERCA-PLB), and three parameters were calculated per interaction (binding affinity, oligomer number, interprobe distance). Results indicate (a) SLN and PLB show high-affinity self-association into homo-oligomers and low-affinity cross-assembly as hetero-dimers, and (b) SERCA forms 1:1 binary complexes with SLN or PLB when co-expressed with either subunit individually, with SERCA having 3-fold higher affinity for SLN over PLB. We conclude that SLN and PLB monomers bind independently to SERCA, and that each subunit shows competing self-association versus regulatory complex formation. Molecular modeling based on FRET parameters was used to examine the SERCA-SLN-PLB complex. We propose that equilibrium allocation of SERCA between binary and ternary complexes depends on the expression level and phosphorylation state of each regulatory subunit in muscle. Acknowledgments: This work was funded by NIH grants to DDT (GM27906, AR0507220, AR007612) and UMN awards to JMA (CBS-HHMI).

We have detected structural dynamics of the sarcoplasmic reticulum Ca-ATPase (SERCA) using time-resolved fluorescence spectroscopy. The Ca-ATPase from fast-twitch skeletal muscle (SERCA1a isoform) was labeled with cyan fluorescent protein (CFP) at the N-terminus in the actuator domain (A) and fluorescein isothiocyanate (FITC) at Lys-515 in the nucleotide-binding domain (N). Time-resolved FRET was detected between CFP (donor in A domain) and FITC (acceptor in N domain) for SERCA in ligand-stabilized states, including calcium-free (E2), calcium-bound (E1), and actively-cycling phosphoenzyme (EP). Lifetime fitting and molecular modeling were used to interpret fluorescence decays, thereby identifying a dynamic distribution of structural states within the cytoplasmic headpiece of SERCA (i.e., A-N interdomain distance). FRET results were compared to distance predictions from x-ray crystallography. We propose a structural mechanism for ligand activation of SERCA. Acknowledgments: Spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Gregory Gillispie, President). This work was funded by NIH grants to DDT (R01 GM27906, P30 AR0507220, T32 AR007612).

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca- ATPase (SERCA) by measuring Forster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a key membrane protein that controls muscle contractility by regulating the calcium transport activity of SERCA, and perhaps by also acting as an ATP-activated anion channel (Becucci et al., Biophys. J. 2007, 2009). Here we used baculovirus expression of fluorescent fusion proteins in insect cells and FRET microscopy to provide novel evidence for (a) independent oligomerization of SLN and (b) regulatory complex formation between SERCA and SLN. FRET assays demonstrated that SLN monomers self-assemble into dimers and higher-order oligomers in the absence of SERCA, but that SLN monomers also bind to SERCA in a 1:1 binary complex when the two proteins are co-expressed. FRET assays further demonstrated that the binding affinity of SLN:SLN homo-oligomers is greater than the binding affinity of SERCA:SLN hetero-dimers, indicating that SLN monomers favor self-association over SERCA binding. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased FRET for SLN self-association and eliminated multimeric assembly of SLN, converting higher-order oligomers into monomers and dimers. The I17A mutation also decreased FRET for SERCA:SLN binding but maintained 1:1 stoichiometry of hetero-dimer formation. Thus, isoleucine-17 is a residue that plays dual roles in (a) determining the distribution of SLN homo-oligomers and (b) stabilizing the formation of SERCA:SLN hetero-dimers. When expressed in bacterial cells, wild-type SLN prevented colony formation but the I17A-SLN mutant did not, indicating that higher-order oligomers of SLN exhibit antibacterial activity, possibly through channel formation. We propose that SLN exists as multiple molecular species in muscle membranes, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (hetero-dimer).

We have performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in complex with phospholamban (PLB). Regulation of SERCA activity by PLB is central in modulating cardiac contractility. X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca2+ affinity E2 state that is incompatible with metal ion binding, thereby blocking the conversion toward a high-Ca2+ affinity E1 state. Estimation of pKa values of the acidic residues in the Ca2+ sites indicates that under normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state deprotonated at residues E309 and D800 yet protonated at residue E771. We performed microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K+ and 3 mM Mg2+. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol, and that two K+ ions bind transiently (≤5 ns), and non-specifically (9 different positions) to the two transport sites; the total occupancy time of the two K+ ions in the transport sites is 80%. These findings indicate that PLB does not inhibit the E2-to-E1 interconversion but instead populates a novel E1 intermediate, E1•H+771, that depresses SERCA activation by Ca2+. We propose that the efficient regulation of SERCA activity by PLB results from structural transitions that occur primarily in the E1 state of the pump. This work was supported by grants to L.M.E-F. from the American Heart Association (12SDG12060656) and to D.D.T from NIH (GM27906), and by the University of Minnesota Supercomputing Institute.

We have detected regulatory interactions between the sarcoplasmic reticulum calcium-transporting ATPase (Ca-ATPase, aka SERCA) and its inhibitory subunit sarcolipin (SLN) from skeletal muscle. SLN inhibits SERCA by uncoupling calcium transport, reducing catalytic efficiency from two to one calcium ion transported per ATP molecule hydrolyzed. Phosphorylation of SLN is the switch that relieves SERCA inhibition; it is unknown whether SLN phosphorylation disrupts the inhibitory complex (dissociation model) or causes a structural rearrangement (subunit model). Crosslinking assays indicate that calcium binding to SERCA disrupts interaction with SLN, suggesting that SLN dissociates once per catalytic cycle during the key step of calcium uncoupling (Toyoshima, Nature 2013). We previously detected oligomeric interactions between the two proteins using fluorescence resonance energy transfer (FRET) microscopy, finding that that SLN binds to SERCA in a 1:1 binary complex in the presence of micromolar calcium and millimolar ATP (Autry, JBC 2011). Here we used FRET spectroscopy to detect regulatory interactions in the presence or absence of calcium and/or ATP, finding that the two proteins show similar binding in calcium-bound and calcium-free states of SERCA, regardless of nucleotide occupancy. We also assayed SLN-SERCA interaction by column chromatography on anion-exchange and nucleotide-mimetic resins; results indicate that SLN remains bound to SERCA following detergent solubilization in the presence of calcium. We conclude that calcium does not dissociate SLN from SERCA, a result consistent with SLN-mediated uncoupling of calcium transport. Addition of calcium/calmodulin-dependent kinase did not disrupt SLN-SERCA interaction; thus, we propose that the non-dissociative subunit model is the molecular mechanism that mediates SLN regulation of SERCA. Acknowledgments: FRET was performed in the Biophysical Spectroscopy Center at the University of Minnesota, with assistance from Fluorescence Innovations, Inc. (Greg Gillispie, President). This work was funded by NIH grants to DDT (R01-GM27906, P30-AR0507220, T32-AR007612).

We have used quantitative immunoblot, enzyme-linked immunosorbant assay (ELISA), and capillary western to measure absolute cardiac sarcoplasmic reticulum Ca2+ ATPase (SERCA2a isoform) content in order to determine specific activity. Celladon, Inc., is developing a gene therapy treatment for heart failure (HF) where SERCA2a is the target protein, and they need an accurate measure of SERCA2a to calculate specific activity of enzyme produced by the gene therapy construct. Patients with HF produce too little SERCA2a or SERCA2a that has too low of activity to maintain normal calcium cycling that is responsible for cardiac contraction. SERCA2a produced by MYDICAR, an adeno- associated viral (AAV) vector, must have comparable activity to endogenous SERCA2a to be useful as a gene therapy product. Immunoblot showed linear SERCA2a detection. We conclude that immunoblot measures absolute SERCA2a content, useful in measuring specific activity. This work was funded by Celladon and by NIH grants to DDT (R01 GM27906, P30 AR0507220, T32 AR007612).

We have monitored molecular interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring Forster resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). SLN regulates contractility in cardiac and skeletal muscle by regulating SERCA calcium uptake and perhaps by acting as an ion channel. Here, live cell FRET microscopy provided novel evidence for self-association of SLN and for regulatory complex formation between SERCA and SLN. Fluorescence photobleaching revealed that SLN self-assembles into dimers but not higher oligomers, and that SERCA and SLN monomers bind together in a 1:1 binary complex. FRET between CFP-SLN:YFP-SLN and CFP-SERCA:YFP-SLN exhibited a hyperbolic dependence on protein concentration, with maximum efficiency of 62 ± 1% and 52 ± 1%, respectively. The concentration of half-maximal FRET was 7.6 ± 0.4 AU for SLN:SLN and 17.0 ± 1.0 AU for SERCA:SLN, demonstrating that the binding affinity of SLN:SLN homo-dimers is 2.2-fold greater than SERCA:SLN hetero-dimers. Mutating SLN residue Ile-17 to Ala (I17A) decreased maximum FRET for SERCA:SLN by 14 ± 4% with no change in binding affinity, indicating that the I17A-SLN mutation causes a structural rearrangement with distance increase of 5 ± 1 A within the binary regulatory complex. Addition of wild-type SERCA (cardiac and fast-twitch isoforms) decreased the binding affinity of the SLN:SLN interaction by 24 ± 6% with no change in maximum FRET, indicating that SLN monomers are in competition between dimerization and SERCA binding. We propose that SLN exists as monomers, homo-dimers, and hetero-dimers in sarcoplasmic reticulum membranes, and that Ile-17 of SLN acts as an allosteric switch in the regulatory complex with SERCA.

We have investigated the structure of phospholamban (PLB) and its regulation of the sarcoplasmic reticulum Ca-ATPase (SERCA) using fluorescence resonance energy transfer (FRET) on fluorescent fusion proteins expressed in living cells. Fusion proteins were created with either a donor fluorophore (cyan fluorescent protein, CFP), or an acceptor fluorophore (yellow fluorescent protein, YFP), attached to one terminus of the protein of interest. Both N-terminal and C-terminal fusions of CFP and YFP were made to SERCA and N-terminal fusions were made to PLB.