doi:10.1016/j.jmb.2010.11.014 J. Mol. Biol. (2011) 405, 707–723

Contents lists available at www.sciencedirect.com Journal of Molecular Biology journal homepage: http://ees.elsevier.com.jmb

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

John Paul Glaves1,2, Catharine A. Trieber1,2, Delaine K. Ceholski1, David L. Stokes3,4 and Howard S. Young1,2⁎

1Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 2National Institute for Nanotechnology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 3Skirball Institute of Biomolecular Medicine, School of Medicine, New York University, New York, NY 10016, USA 4New York Structural Biology Center, New York, NY 10027, USA

Received 19 July 2010; Phospholamban physically interacts with the sarcoplasmic reticulum received in revised form calcium pump (SERCA) and regulates contractility of the heart in response 2 November 2010; to adrenergic stimuli. We studied this interaction using electron microscopy accepted 8 November 2010 of 2D crystals of SERCA in complex with phospholamban. In earlier studies, Available online phospholamban oligomers were found interspersed between SERCA dimer 23 November 2010 ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the Edited by W. Baumeister effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps Keywords: from negatively stained and frozen-hydrated crystals, phosphorylation of SERCA; Ser16 selectively disordered the cytoplasmic domain of wild type phospho- phospholamban; lamban. This was not the case for a pentameric gain-of-function mutant phosphorylation; (Lys27Ala), which retained inhibitory activity and remained ordered in the electron crystallography; phosphorylated state. A partial loss-of-function mutation that altered the 2D crystals 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. © 2010 Elsevier Ltd. All rights reserved.

Introduction

*Corresponding author. E-mail address: Cation transport by the P-type ion pumps is an [email protected]. essential process in all eukaryotic cells, where Abbreviations used: SERCA, sarcoplasmic reticulum changes in intracellular cation concentrations are calcium ATPase; SR, sarcoplasmic reticulum; PLB, linked to precise physiological responses. The best phospholamban; PKA, cAMP-dependent protein kinase. understood members of this transport family include

0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved. 708 Co-crystals of SERCA and Phospholamban the sarcoplasmic reticulum calcium ATPase binds to and inhibits SERCA, and phosphorylation (SERCA) found in muscle cells and the plasma disrupts this inhibitory complex.24,25 However, membrane sodium-potassium ATPase (Na+-K+ there is contradictory evidence about whether PLB pump) found in all cell types. These two P-type ion is physically dissociated from SERCA following pumps are particularly important in cardiac con- phosphorylation. Fluorescence energy transfer tractility and are major drug targets for the clinical experiments suggest that PLB inhibits and aggre- improvement of heart disease. An extensive series of gates SERCA, and that phosphorylation reverses X-ray and electron crystallographic studies have this process and causes dissociation of PLB and resulted in structures of a variety of SERCA reaction SERCA.26 Similarly, cross-linking experiments in- intermediates, thus revealing how ATP hydrolysis is dicate that phosphorylation weakens the physical coupled to calcium transport across the sarcoplasmic association of PLB with SERCA and makes the recticulum (SR) membrane in order to achieve complex more susceptible to dissociation by – muscle relaxation.1 14 These studies show that sub-saturating concentrations of calcium.27,28 In SERCA is composed of a transmembrane domain contrast, studies using co-immunoprecipitation,29 that contains the calcium-binding sites and three fluorescence30,31 and EPR spectroscopy32 all sug- cytosolic domains that are responsible for nucleotide gest that PLB remains associated with SERCA binding, phosphorylation, and communication with following phosphorylation. Rather than dissocia- the transmembrane domain. It has been shown that tion of PLB from SERCA after phosphorylation, the intermediate states (calcium binding, phosphor- EPR and NMR studies point to a transition from ylation, calcium transport, dephosphorylation and order to disorder in the cytoplasmic domain of proton counter-transport) involve coupled domain PLB.32,33 Such a transition is consistent with the movements that link the cation-binding sites with results of a variety of biophysical studies showing the phosphorylation state of the enzyme. that phosphorylation causes a partial unwinding Despite this wealth of structural information, the and disordering of the PLB N-terminal α-helix – regulation of the calcium pump in cardiac muscle around the Ser16 phosphorylation site.34 38 Regu- remains an elusive target of study. In cardiac and lation by phosphorylation is thought to occur in the smooth muscle, SERCA is regulated by phospho- context of a complex between monomeric PLB and lamban (PLB), a 52 residue integral membrane SERCA yet it has been suggested that the PLB protein. PLB engages in an inhibitory interaction pentamer is necessary for regulation of cardiac with SERCA that reduces its apparent calcium contractility in a physiological context;39 and a affinity. This is a dynamic process that depends on direct interaction has been proposed between the – the cytosolic calcium concentration, as well as the PLB pentamer and SERCA.40 42 EPR measurements phosphorylation and oligomeric states of PLB, which of boundary lipids suggest that phosphorylation of is in dynamic equilibrium between monomeric and PLB shifts the population towards the oligomeric homo-oligomeric states, with pentameric forms being state.16 These results raise questions about the role dominant in SDS-PAGE.15,16 Mutation of key leucine of PLB oligomeric states in the regulation of SERCA. and isoleucine residues in the transmembrane do- In earlier work, we observed a direct interaction main of PLB destabilizes the pentameric structure between an oligomeric form of PLB and SERCA in 2D and has been shown to shift this equilibrium in favor crystals.42 Specifically, we characterized co-crystals of of the monomer. These pentamer-disrupting muta- SERCA and a super-inhibitory mutant of PLB tions are associated with increased inhibition of (Ile40Ala; I40A).42 While SDS-PAGE indicated that SERCA, leading to the speculation that the PLB this mutant of PLB was monomeric,17 our projection – monomer is the active inhibitory species,17 19 and map revealed that I40A formed an oligomer, which that the pentamer is an inactive storage form.20,21 was later supported by fluorescence resonance energy Unfortunately, it has not been possible to test this transfer experiments.43,44 These results reinforce the model directly with well defined PLB oligomeric conclusion reached by Jones and co-workers, that states within a lipid membrane. In any case, SERCA SDS-PAGE might indicate the relative stability of PLB inhibition by the PLB monomer can be reversed either oligomers but it is not a definitive means of assessing by elevated cytoplasmic calcium concentrations or by the oligomeric species adopted by PLB within the phosphorylation of PLB. The primary physiological lipid bilayer.18 A 3D model based on our projection mechanism for relieving SERCA inhibition is through map suggested that PLB pentamers interact with the phosphorylation of PLB at Ser16 by cAMP- SERCA at two potential sites; one near transmem- dependent protein kinase (PKA), and PLB can be brane segment M3 and another near the C-terminus. phosphorylated at Thr17 by either calcium/calmod- These contact sites are distinct from the inhibitory site ulin-dependent protein kinase II22 or by Akt.23 occupied by the PLB monomer, which is adjacent to The functional effect of PLB phosphorylation on M2, M4 and M6 of SERCA according to the results of – SERCA regulation is clear but the mechanism for mutagenesis and cross-linking studies.45 49 In this this effect is less certain. The original model for study, we investigated the effects of PLB phosphory- SERCA regulation suggested that monomeric PLB lation and mutation on the interaction between a PLB Co-crystals of SERCA and Phospholamban 709

oligomer and SERCA in the context of 2D crystals. Table 1. Apparent calcium affinity (KCa) determined for Our results show a correlation between PLB function SERCA in the absence and in the presence of wild type, and crystal formation, suggesting that the physical mutant and phosphorylated forms of PLB interactions that stabilize the crystal are sensitive to μ Δ KCa ( M calcium) KCa physiologically relevant perturbations. Our data are – consistent with an order-to-disorder transition in the SERCA (n=28) 0.41±0.01 PLBwt (n=9) 0.69±0.01 0.28 PLB cytoplasmic domain, where the inhibitory forms Phospho-PLBwt (n=3) 0.43±0.02 0.02 of PLB (e.g. wild type and the gain-of-function mutant K27A (n=7) 1.00±0.03 0.59 Lys27Ala) retain an ordered state in the crystals and Phospho-K27A (n=3) 0.65±0.03 0.24 the non-inhibitory forms of PLB (phosphorylated wild R14A (n=3) 0.59±0.03 0.18 N34A (n=6) 0.46±0.03 0.05 type and a loss-of-function mutant Asn34Ala) adopt a Δ disordered state. KCa is the change in calcium concentration at half-maximal ATPase activity of SERCA in the presence of the wild type, mutant and phosphorylated forms of PLB; calculated as the Results difference in KCa values for SERCA in the absence and in the presence of PLB.

Co-reconstitution of SERCA and PLB 2b) and matrix-associated laser desorption ionization Methods for co-reconstituting SERCA and PLB time-of-flight mass spectrometry (MALDI-TOF; data have been established for both functional50,51 and not shown) were used to demonstrate stoichiometric structural studies.42,52,53 Co-reconstituted proteoli- phosphorylation of wild type and K27A PLB. The posomes have been shown to have a lipid/protein R14A and the N34A mutants were not treated with molar ratio of approximately 120:1 and a PLB/ PKA because the former is not recognized by PKA56 SERCA molar ratio of 3.5:1.42,51 These conditions and the latter (loss-of-function) is not affected by mimic cardiac SR and, under the appropriate buffer phosphorylation. Finally, our observation that phos- conditions, they promote formation of 2D crystals. phorylated K27A remains partially inhibitory (Fig. Measurements of ATPase activity have been used to 2a) is consistent with earlier work demonstrating that demonstrate the regulatory interactions between phosphorylated N27C also retains its inhibitory wild type and mutants forms of PLB and SERCA. capacity and can be cross-linked to SERCA.28 Note For the current studies, wild-type PLB, a gain- that residue 27 is lysine in the human protein (used of-function mutant (Lys27Ala), a partial loss-of- here) and asparagine in the canine protein.28 function mutant (Arg14Ala), and a complete loss-of- function mutant (Asn34Ala) were chosen to repre- Co-crystallization of SERCA and PLB – sent different functional forms of PLB.17,19,51,53 55 Importantly, the oligomeric stability of the mutants We next tested the ability of wild type, mutant and used for 2D crystallization was similar to that of phosphorylated forms of PLB to interact with wild type PLB, despite differences in their ability to SERCA in large 2D co-crystals.42 The proteolipo- regulate SERCA.17,55 Measurements of ATPase somes described above were capable of forming activity were used to determine the apparent crystals after treatment with decavanadate, EGTA, calcium affinity of SERCA in the absence and in and a freeze-thaw procedure designed to enhance the presence of PLB (Table 1). Reconstituted SERCA proteoliposome fusion and crystal growth. Howev- in the absence of PLB had a KCa value of 0.41± er, the PLB mutants varied markedly in their ability 0.01 μM calcium, and co-reconstituted SERCA in the to form co-crystals. In order to characterize the effect presence of wild-type PLB had a KCa value of 0.69± of mutation and phosphorylation on crystal forma- μ μ 0.01 M calcium. The KCa values ( M calcium) in the tion, care was taken to ensure that the crystallization presence of mutant forms of PLB were 1.00±0.03 for conditions were equivalent between the various K27A, 0.59±0.03 for R14A and 0.46±0.03 for N34A. samples. The number of crystals in a grid square We tested the effect of PKA phosphorylation on the (2500 μm2) was counted in negatively stained inhibitory capacity of PLB. After co-reconstitution, samples (Table 2). Wild type PLB produced a phosphorylation of wild type PLB restored the moderate frequency of about three crystals in a apparent calcium affinity of SERCA to control levels grid square and other samples revealed a correlation (Fig. 1a; Table 1). However, phosphorylation of the between the functional state of PLB and its propen- K27A mutant by PKA did not completely restore sity to form co-crystals with SERCA. For example, the apparent calcium affinity of SERCA (Fig. 2a; Table the K27A gain-of-function mutant formed about five 1). The KCa values in the presence of phosphorylated crystals in a grid square, and the N34A loss-of- forms of PLB were 0.43±0.02 μM calcium for wild function mutant formed about one crystal in a grid type (nearly complete (93%) reversal of inhibition) square. This analysis was not done for the R14A and 0.65±0.03 μM calcium for K27A (partial (59%) mutant. Despite changes in crystal frequency and reversal of inhibition). Both SDS-PAGE (Figs 1b and order, the crystal morphology and lattice parameters 710 Co-crystals of SERCA and Phospholamban

Fig. 1. Co-reconstitution and co-crystallization of SERCA with non-phosphorylated and phosphorylated wild type PLB. (a) ATPase activity of SERCA reconstituted in the absence (●) and in the presence of wild type (▼) and ◼ phosphorylated wild type ( ) PLB. The calcium affinity values (KCa) are reported in the text, and all curves have been normalized to the maximal activity (V/Vmax). (b) An example of staining with Coomassie brilliant blue after SDS-PAGE of the co-reconstituted proteoliposomes used in the crystallization studies. SERCA and the wild type PLB pentamer (PLB5) are labeled. Co-reconstituted proteoliposomes were run on 10% (top) and 16% (bottom) polyacrylamide gels. A fivefold larger amount of sample was loaded onto the 16% gel for display purposes. A characteristic shift was observed in the mobility of the PLB pentamer after phosphorylation (ph-PLB5) with protein kinase A (PKA). (c) Projection map of negatively stained co-crystals of SERCA in the presence of wild type PLB. A single unit cell (a≈345 Å, b≈70 Å) and symmetry operators are indicated for the p22121 plane group. The green densities indicate a single SERCA molecule, where the negative stain reveals only the cytoplasmic domain. The relative locations of the actuator (A) and nucleotide- binding (N) domains are indicated. The densities associated with PLB are interspersed between the SERCA dimer ribbons. (d) The projection map from negatively stained co-crystals of SERCA in the presence of phosphorylated wild-type PLB. The projection maps (c and d) are contoured showing all negative (b0; broken lines) and positive (≥0; continuous lines) densities; each contour level corresponds to 0.25 σ. were similar to one another and to those reported averaging Fourier data from five different crystal 42 earlier. In particular, all crystals exhibited p22121 images at a resolution of ~20 Å. To rule out plane group symmetry with approximate lattice differences in negative staining, two or three dimensions of a=345 Å and b=70Å (γ=90°). projection maps were calculated for each form of PLB, where each map represented an independent Projection maps from negatively stained co-reconstitution, crystallization and negative stain 2D co-crystals EM grid. Typical projection maps are shown for non-phosphorylated and phosphorylated wild type For each form of PLB, projection maps of PLB (Fig. 1). Consistent with what was observed negatively stained samples were calculated after earlier,42 the projection maps were dominated by Co-crystals of SERCA and Phospholamban 711

Fig. 2. Co-reconstitution and co-crystallization of SERCA with non-phosphorylated and phosphorylated K27A PLB. (a) ATPase activity of SERCA reconstituted in the absence (●) and in the presence of K27A (▼) and phosphorylated K27A ◼ ( ) PLB. The calcium affinity values (KCa) are given in the text, and all curves have been normalized to the maximal activity (V/Vmax). (b) An example of a Coomassie brilliant blue-stained SDS-PAGE of the co-reconstituted proteoliposomes used in the crystallization studies. SERCA and the K27A PLB pentamer (PLB5) are labeled. Co- reconstituted proteoliposomes were run on 10% (top) and 16% (bottom) polyacrylamide gels. A fivefold greater amount of sample was loaded onto the 16% gel for display purposes. A characteristic shift was observed in the mobility of the PLB pentamer after phosphorylation (ph-PLB5) with PKA. (c) Projection map from negatively stained co-crystals of SERCA in the presence of K27A PLB. (d) Projection map from negatively stained co-crystals of SERCA in the presence of phosphorylated K27A PLB. The projection maps (c and d) are contoured showing only positive (continuous lines) densities; each contour level corresponds to 0.25 σ.

Table 2. Lattice parameters and crystal propensity for negatively stained crystals of SERCA with and without phospholamban

p22121 lattice parameters Characteristics abγ Crystal frequency Crystal quality PLB Pentamer, inhibitory 341.3±2.7 70.3±0.3 90.2±0.5 3.1±0.5 (n=7) Intermediate Phospho-PLB Pentamer, non-inhibitory 341.6±6.2 70.2±0.9 90.1±1.0 1.4±0.4 (n=7) Poor K27A Pentamer, gain-of-function 344.9±4.2 70.9±0.9 85.6±1.5 5±1 (n=6) High Phospho-K27A Pentamer, inhibitory 339.5±1.5 71.3±0.5 90.1±0.4 1.7±0.5 (n=7) Poor to intermediate N34A Pentamer, loss-of-function 339.1±2.0 70.6±0.7 89.5±0.5 1±0.4 (n=7) Poor Crystal frequency is the average number of crystals observed per grid square (400-mesh grids) for a minimum of six independent co- reconstitutions and crystallization trials. This was not done for the R14A mutant of PLB. For each independent co-reconstitution and crystallization trial (n), at least 30 grid squares were examined for crystal frequency. 712 Co-crystals of SERCA and Phospholamban

Fig. 3. Co-reconstitution and co-crystallization of SERCA with R14A PLB. (a) ATPase activity of SERCA reconstituted ● ▼ in the absence ( ) and presence of R14A ( ) PLB. The calcium affinity values (KCa) are given in the text, and all curves have been normalized to the maximal activity (V/Vmax). (b) The projection map from negatively stained co-crystals of SERCA in the presence of R14A PLB. The projection map is contoured showing only positive (continuous lines) densities; each contour level corresponds to 0.25 σ. rows of 2-fold related densities that corresponded to phosphorylation of K27A had no effect on the PLB anti-parallel dimer arrays of SERCA. In the presence density (compare Figs. 1d and 2d). This behavior is of wild type PLB, additional density was inter- consistent with the fact that stoichiometric phos- spersed between the SERCA arrays, consistent with phorylation of K27A is unable to fully reverse its the presence of PLB oligomers (Fig. 1c). These inhibition of SERCA (Fig. 2a and b). We reasoned patches of extra density were relatively small in that if phosphorylation disrupted a crystal contact the maps of negatively stained crystals, owing to the or altered contrast produced by negative stain (e.g., low contrast generated by negative stain within the by adding a negative charge to the cytoplasmic lipid bilayer and the small size of the cytoplasmic domain), then phosphorylation of K27A co-crystals domain of PLB relative to SERCA. Nonetheless, the should produce a result similar to the wild type PLB PLB density in the projection maps was similar to (Fig. 1c and d). However, if co-crystallization relies those reported earlier.42 Upon phosphorylation of on a functional interaction between PLB and Ser16 with PKA, the PLB density was no longer SERCA, then the inhibitory properties of the present, consistent with disordering of the cytoplas- phosphorylated K27A mutant should correlate mic domain (Fig. 1d). As mentioned above, phos- with its behavior during co-crystallization. Based phorylation of wild type PLB also reduced the on our observations, we believe that the cytoplasmic frequency of co-crystal formation (Table 2). domain of wild type PLB becomes disordered upon We studied the effect of phosphorylation on the phosphorylation, whereas the cytoplasmic domain co-crystals with K27A PLB, which has the same of K27A does not. This comparison rules out the oligomeric state as wild type PLB.17,55 Like wild possibility that the disappearance of the PLB density type PLB, the projection maps of non-phosphory- was a result of simply adding the negative charge 2– lated K27A co-crystals show PLB density lying (PO4 ) to its cytoplasmic domain. between the anti-parallel dimer ribbons of SERCA (Fig. 2). The frequency of the K27A co-crystals is Additional mutants of PLB higher than that of the wild type, reflecting the fact that the K27A mutation produces a super-inhibitory Since the phosphorylation of wild type PLB PLB molecule (gain of function). Interestingly, reverses SERCA inhibition and alters density in the Co-crystals of SERCA and Phospholamban 713

Fig. 4. Co-reconstitution and co-crystallization of SERCA with N34A PLB. (a) ATPase activity of SERCA reconstituted ● ▼ in the absence ( ) and in the presence of N34A ( ) PLB. The calcium affinity values (KCa) are given in the text, and all curves have been normalized to the maximal activity (V/Vmax). (b) Projection map from negatively stained co-crystals of SERCA in the presence of N34A PLB. The projection map is contoured showing all negative (broken lines) and positive (continuous lines) densities; each contour level corresponds to 0.25 σ.

2D co-crystals, we tested the effects of (i) mutation of a SERCA was negatively impacted by PLB phospho- charged residue (Arg14) proximal to the phosphory- rylation and that the cytoplasmic domain of PLB lation site (Ser16) and (ii) a well-characterized loss-of- tended to become disordered. To test if this function mutant (N34A). As reported earlier,17,56 R14A disordering affected the transmembrane domain retains substantial inhibitory capacity (Fig. 3a), where- of PLB, we imaged the co-crystals in the frozen- as N34A is a complete loss-of-function mutant (Fig. 4a). hydrated, unstained state. Based on molecular The R14A mutant changes the net charge of the PLB models for PLB,41,57,58 the density associated with cytoplasmic domain adjacent to the phosphorylated PLB in projection maps from frozen-hydrated co- residue Ser16, while the N34A mutant is located at the crystals is dominated by the pentameric transmem- interfacebetweenthemembraneandthecytosol.In brane coiled coil. This is due to the alignment of the projection maps of negatively stained co-crystals with transmembrane helices along the imaging direction, R14A PLB, additional density was observed between which produces very strong density in the the rows of SERCA molecules (Fig. 3b). However, in corresponding projection maps. Therefore, compar- co-crystals with N34A PLB, the additional density was ison of the density observed in negatively stained absent (Fig. 4b). These results further support the idea co-crystals with that observed in frozen-hydrated that the functional state, rather than the net charge of crystals allows us to evaluate the relative ordering the cytoplasmic domain, determines the stability of of the cytoplasmic and transmembrane domains, co-crystals and whether PLB-associated densities are respectively. In particular, if phosphorylation dis- visible in the projection maps. Interestingly, the N34A orders primarily the cytoplasmic domain of PLB as mutation appears to cause a disordering of the predicted,32 density attributable to PLB should be cytoplasmic domain that is similar to the effect of weak in the negatively stained samples and phosphorylation of the wild type PLB. unaffected in frozen-hydrated samples. For this comparison, projection maps were calculated from Projection maps from frozen-hydrated images of frozen-hydrated co-crystals with wild 2D co-crystals type PLB before and after PKA phosphorylation. Following merging and averaging of data from at Our observations of negatively stained crystals least five crystal images, diffraction amplitudes indicated that the co-crystallization of PLB and with high signal-to-noise ratios and low phase 714 Co-crystals of SERCA and Phospholamban

Table 3. Summary of crystallographic data for frozen- additional interactions in the non-phosphorylated hydrated co-crystals of SERCA and phospholamban state. Phospholamban a What is the oligomeric state of PLB in the I40A K27A co-crystals? Number of images 5 15 Cell parameters To further characterize the physical interaction a (Å) 359.2 346.5 b (Å) 71.9 70.7 between SERCA and PLB and to evaluate its γ (°) 90.3 89.7 oligomeric state in the co-crystals, we used frozen- Overall weighted phase residual (°)b 16.8 16.1 hydrated preparations to improve the resolution of 42 a Data from Stokes et al. (2006).42 the existing projection map. We chose to image co- b Including data to IQ 7. crystals of K27A PLB because the relative abun- dance and high quality of these crystals facilitated data collection. Images from frozen-hydrated co- crystals displayed computed diffraction to a resolu- residuals were observed for all resolution shells to a tion of approximately 15 Å. Following merging and resolution of 10 Å (Table 3). The resulting maps averaging of data from 15 crystal images, diffraction were nearly identical, both containing anti-parallel amplitudes with high signal-to-noise ratios and low dimer ribbons of SERCA molecules interspersed phase residuals were observed for all resolution with density consistent with pentameric PLB (Fig. 5). shells to a resolution of 8 Å (Fig. 6; Table 3). This similarity suggests that intramembrane inter- The resulting projection map for SERCA in the actions between SERCA and the transmembrane presence of K27A PLB is similar to that deter- domain of PLB mediate contacts in the crystals mined for SERCA in the presence of I40A PLB.42 and that these contacts persist after phosphoryla- The size and shape of the additional densities tion of the PLB cytoplasmic domain. Loss of seen in our projection maps were consistent with density for these cytoplasmic domains suggests the pentamer, which is the principle oligomeric that they are disordered and thus not strongly form of PLB observed by SDS-PAGE. However, bound to SERCA after phosphorylation. Given the neither map from frozen-hydrated co-crystals negative effect of phosphorylation on crystal order, (I40A reported earlier42 or K27A reported here) the PLB cytoplasmic domain probably provides provided direct evidence of pentameric assembly

Fig. 5. Projection maps from frozen-hydrated co-crystals of SERCA in the presence of (a) wild type and (b) phosphorylated wild type PLB. Statistics for merging five crystal images indicated that phase residuals were b35° to a resolution of approximately 10 Å. The region of the map shown is approximately 600 Å×159 Å. The relative locations and orientations of SERCA molecules are indicated by arrows and the locations of the PLB densities are indicated by brackets. The projection maps are contoured showing only positive (continuous lines) densities. Co-crystals of SERCA and Phospholamban 715

Fig. 6. Projection map from frozen-hydrated co-crystals of SERCA in the presence of K27A PLB. (a) Statistics for merging 15 crystal images indicate that phase residuals are b26° to a resolution of 8 Å. (b) Projection map generated for co-crystals of SERCA and K27A PLB. The region of the map shown is approximately 600 Å×159 Å. The PLB densities are similar to those characterized for I40A PLB,42 reflecting an identical oligomeric state and mode of interaction with SERCA. The projection map is contoured showing only positive (continuous lines) densities. of PLB, presumably due to limited resolution. To Discussion address this issue, the high-resolution terms of the projection map were enhanced by applying anegativeB-factor (temperature factor) of Physical interactions between SERCA and PLB – 500 Å 2.59 The truncation of Fourier data at 8 Å resolution ensured that the contribution of noise There is general consensus that the monomeric in our map was minimal. This procedure im- form of PLB interacts with M2, M4 and M6 of proved contrast and detail for the densities SERCA,17,19,20,25,48,49 producing the inhibition that associated with both SERCA and PLB (Fig. 7). characterizes the resting state of cardiac muscle. Significantly, the density assigned to the PLB However, oligomeric forms of PLB have been oligomer resolved into a five-lobed density con- observed repeatedly both in detergent and mem- sistent with the PLB pentamer. However, rather branous environments. While the pentamer appears than the symmetric structure predicted by NMR to be the most stable oligomer, tetramers, trimers structural models,41,58 our density resembles a and dimers have all been observed and the balance distorted pentamer (Fig. 7b–d), where two of the between them can be influenced by single-site five observed lobes are stronger and better mutations.17,19 Furthermore, there is dynamic ex- delineated than the others. Although the shape change between oligomeric and monomeric forms of of the pentamer might be affected by the PLB and evidence that phosphorylation of PLB or increased noise in the “sharpened” projection increased cytosolic calcium concentrations increase map, a physical distortion of the pentamer the proportion of the pentameric pool at the expense would be consistent with its asymmetric interac- of the monomeric pool.16 This evidence has led to tion with SERCA. Interestingly, the stronger the hypothesis that the monomer represents the densities in the pentamer are proximal to “active” inhibitory form and that oligomeric forms SERCA, and one appears next to transmembrane represent an inactive reserve.20,21 While several segment M3 (asterisk in Fig. 7a). An interaction congruent molecular models explaining the inhibi- between M3 of SERCA and PLB was also tory properties of PLB are described in the suggested by our earlier studies of the complex literature,47,48,60,61 there are a number of inconsis- using electron cryo-microscopy of helical crystals tencies in published observations that are not (Fig. 4 in 53). fully explained. First, Kranias and colleagues 716 Co-crystals of SERCA and Phospholamban

Fig. 7. The projection map recalculated with an applied negative B-factor. (a) In the sharpened projection map, the contrast and level of detail is enhanced for both SERCA and PLB. The asterisk (⁎) indicates the region of closest contact between the PLB pentamer and transmembrane segment M3 of SERCA. (b) A close up view of the density associated with PLB. The size and shape of the PLB densities are now compatible with a pentamer. (c) A simulated projection for a transmembrane pentamer is shown for comparison. (d) Superimposition of the maps shown in (b) and (c), indicating a slightly distorted pentameric arrangement. demonstrated that a mutant form of PLB (Cys41Phe) observations on how the physical association was insufficient for proper regulation of cardiac between SERCA and PLB responds to phosphory- contractility in a mouse model.39 Because this lation. One group of studies postulate that PLB mutant was reported to be monomeric with the remains associated with SERCA and phosphoryla- same inhibitory potency as the wild type protein,62 tion alters the structural interaction between the two – the authors inferred a physiological role for the PLB proteins,29,30 32,64 whereas contradictory crosslink- pentamer. Despite the fact that the oligomeric state ing studies have led to the notion that intermolec- of the C41F mutant has not been demonstrated ular interactions at the M2, M4 and M6 interface are directly in a membranous environment, this notion lost following phosphorylation27,28 and PLB dis- is consistent with independent observations that sociates from SERCA. SERCA might interact with oligomeric forms of More specifically, chemical crosslinking experi- – PLB.40 42,63 Thus, the existence of oligomers appears ments indicated that PKA-mediated phosphoryla- to offer a functional advantage for the SERCA–PLB tion decreases the efficiency of crosslinking to interaction,40 and this advantage is not explained by SERCA at multiple sites distributed throughout current molecular models (i.e., PLB oligomers as both the cytoplasmic and the transmembrane inactive storage forms). Second, there are disparate domains of PLB.27,28 The inference then is that Co-crystals of SERCA and Phospholamban 717 phosphorylation decreases binding affinity of PLB for govern the association of the monomer with the SERCA and causes it to dissociate at sub-saturating inhibitory site of SERCA are directly related to those concentrations of calcium. Contradictory evidence used in the interaction of a PLB oligomer at the in favor of persistent association comes from the secondary, accessory site. following literature. Antibodies recognizing PLB phosphorylated at Ser16 were shown to co- immunoprecipitate SERCA1a and SERCA2a after Effect of phosphorylation on physical co-expression in HEK-293 cells.29 Fluorescence, spin- interaction between SERCA and PLB label EPR spectroscopy and other biophysical studies demonstrated that SERCA restricts the In studies of the PLB monomer, there is some motional freedom of PLB both before and after consistent evidence that phosphorylation of Ser16 phosphorylation.30,31 More recent EPR studies also causes localized changes in the structure of its support persistent association and suggest that cytoplasmic domain.32,33,35,65,66 These results pro- phosphorylation induces a dynamically disordered vide a physical basis for disruption of the productive state in the PLB cytoplasmic domain and supported structural interaction between SERCA and PLB, the idea that phosphorylated PLB remains associated which might lead to dissociation of the complex. with SERCA in a non-inhibitory state.32 Despite this Early NMR and CD spectroscopy studies showed a growing body of evidence, it is difficult to envision continuous α-helix in the N-terminal portion of PLB how PLB might remain bound to the M2, M4 and M6 (residues 1–16), which partially unwound upon interface of SERCA, given the large conformational phosphorylation (residues 12–16 were no longer changes caused by calcium binding that occlude this helical).35 However, these studies utilized an interaction interface. N-terminal fragment of PLB (residues 1–25), rather These apparent contradictions can be reconciled than the full-length protein, and did not include by hypothesizing a physical interaction between SERCA. Since then, there have been many struc- SERCA and oligomeric PLB at a secondary, non- tural models for full-length PLB based on NMR – inhibitory site. Specifically, we have observed an measurements in the non-phosphorylated,41,61,67 71 interaction between M3 of SERCA and the PLB phosphorylated65 and pseudo-phosphorylated72 pentamer in the 2D co-crystals. Such an interaction state under a variety of experimental conditions. could explain the mutual effects that SERCA and These models differ in the amount of secondary PLB have on spectroscopic analysis of each other's structure in the N-terminal cytoplasmic domain of aggregation state42,63 and could provide a direct role PLB, suggesting that this domain can adopt a variety for the PLB oligomer under physiological of conformational states. Furthermore, most studies conditions.39 In addition, distinct functional con- agree that phosphorylation alters the dynamics sequences and binding sites of the monomer and of this domain, the NMR structure of a pseudo- oligomer could explain the experimental discrepan- phosphorylated form of PLB notwithstanding.72 cy regarding the persistence of the interaction. The A variety of biophysical studies indicate that PLB monomer might dissociate from the M2, M4 PLB undergoes a conformational change upon and M6 inhibitory site under the appropriate phosphorylation.12,34,35,73 Many of these ideas coa- physiological stimuli and there could be a high lesced in recent EPR32 and NMR33,66 studies, which probability for interaction with the PLB oligomer at proposed an order-to-disorder transition in the the secondary site on the other side of the SERCA cytoplasmic domain of PLB that disrupts intermo- molecule (e.g., M3). Compared to the dramatic lecular contacts and reverses SERCA inhibition, but movements of M2, M4 and M6 during calcium does not dissociate the SERCA–PLB complex. binding, the transmembrane helix M3 of SERCA is Consistent with this idea, we found that phos- less mobile and might represent a stable interaction phorylation of wild type PLB at Ser16 selectively point for a PLB oligomer that is insensitive to disordered the cytoplasmic domains of the penta- phosphorylation or the level of cytosolic calcium. mer (Fig. 1) and reduced its ability to mediate 2D In the present work, our data support a function- crystallization with SERCA (Table 2). In maps from ally relevant interaction between SERCA and a PLB negatively stained crystals, density attributable to oligomer in 2D co-crystals. In particular, the effects the cytoplasmic domain disappeared when PLB was of PLB mutants on crystallization are correlated phosphorylated, whereas maps from frozen-hydrat- with their effects on the inhibition of SERCA. The ed crystals showed that the transmembrane helices K27A and N34A mutations were chosen for the of PLB were still clearly visible. We conclude that the similar stability of their pentameric form, yet widely transmembrane domain of phosphorylated PLB different inhibitory behavior. Thus, it is significant remains associated with SERCA in the co-crystals, that the gain-of-function K27A mutation enhanced even though the cytoplasmic domain became crystallization, whereas the loss-of-function N34A disordered. This disordering did not occur for the mutation interfered with crystallization. We con- K27A gain-of-function mutant (Fig. 2) but it did clude that the structural properties of PLB that occur for the N34A loss-of-function mutant, even in 718 Co-crystals of SERCA and Phospholamban the non-phosphorylated state (Fig. 4). Furthermore, the propensity to induce co-crystallization was retained by the K27A mutant after phosphorylation, whereas the N34A mutant was marginal in co- crystallization, even in the non-phosphorylated state (Table 2). It is interesting to consider where these three residues lie in the structure of PLB. Ser16 flanks the N-terminal end of domain Ib, Lys27 is found in the middle of this domain and Asn34 flanks the C-terminal end. These residues also have two opposed functional effects; PLB function is lost when phosphorylated at Ser16 or mutated at Asn34 and PLB function is enhanced when mutated at Lys27. Thus, domain Ib might be the key structural element responsible for phosphorylation- or mutation-dependent conformational changes in the PLB cytoplasmic domain that impact SERCA inhibition. Indeed, many of the structural differ- ences between existing PLB models involve – domain Ib,41,61,65,67 72 suggesting that this domain might be a less structured, more dynamic region of the protein. Therefore, it is reasonable to suppose that phosphorylation alters the dynamics of domain Ib, thereby controlling an order-to- disorder transition in the PLB cytoplasmic domain and reversing SERCA inhibition.29,31,32

Model for the interaction of the PLB pentamer

The major findings in this study are that the PLB pentamer is capable of a physical interaction with SERCA and that this interaction is sensitive to functional modification of PLB through phospho- rylation or mutation. In particular, PKA-mediated phosphorylation of Ser16 caused the cytoplasmic domain of PLB to become disordered, yet the Fig. 8. A partial sequence alignment and a diagram for pentamer remained associated with SERCA through the interaction of PLB with SERCA. Upper: The potential intramembrane interactions. This disordering oc- sequence similarity between the transmembrane domain curred also for a well characterized loss-of-function of PLB (residues 32–52) and transmembrane segment M3 mutation, Asn34Ala, suggesting that the loss of of SERCA (residues 254–274). Leu44, Ile47 and Leu51 make inhibition might occur through a molecular mech- up part of the leucine-isoleucine zipper that stabilizes the anism similar to phosphorylation. These data are pentameric state of PLB. Lower: The pentamer and consistent with a functional interaction between monomer are in dynamic equilibrium, where the monomer SERCA and PLB oligomers,40 and inconsistent with is postulated to interact with and inhibit SERCA (right- the notion of PLB oligomers as inactive storage hand side). We hypothesize that the pentamer also forms.21 The inhibitory site involving the PLB interacts with SERCA, which leads to the active association or dissociation of a monomer (left-hand side). The active monomer and transmembrane helices M2, M4 and 45,48,61 dissociation of a monomer leads to a physical interaction M6 of SERCA is distinct from the physical with and inhibition of SERCA and this process is reversed interactions that stabilize our 2D crystals, which by phosphorylation of PLB. These two pathways are not center on M3 of SERCA and involve the transmem- mutually exclusive and might operate simultaneously or brane domain of PLB. The inhibitory site of SERCA under disparate physiological conditions. alternately opens and closes during the calcium transport cycle, owing to large movements of transmembrane helices M2, M4 and M6. This is not the case for the accessory site of SERCA, because comparison between SERCA and PLB reveals a M3 is less mobile during the transport cycle. Thus, region of potential sequence similarity that spans the M3 could act as an interaction point between SERCA C-terminal portion of the PLB transmembrane helix and the PLB pentamer (and perhaps other oligo- and M3 of SERCA (Fig. 8). Residues Leu266, Val269 meric forms). Interestingly, a primary structure and Leu273 of SERCA face the lipid environment Co-crystals of SERCA and Phospholamban 719 and adopt a side chain orientation similar to that of Reconstitution of SERCA with PLB Leu44, Ile47 and Leu51 of PLB. These residues form part of the leucine-isoleucine zipper that stabilizes SERCA was prepared from rabbit hind leg muscle76 by 9 the pentameric form of PLB.74 affinity chromatography. Recombinant human PLB was 77 Through its interaction with M3 of SERCA, the prepared as described. The following proteins were used in PLB pentamer might play a more active role in this study: wild type PLB, a gain-of-function mutant capturing monomeric or phosphorylated PLB spe- Lys27Ala (K27A), a partial loss-of-function mutant Arg14Ala – (R14A), and a loss-of-function mutant Asn34Ala (N34A). Co- cies. Specifically, this SERCA pentamer interaction reconstitution of SERCA with PLB followed established might facilitate the exchange of PLB monomers methods for the formation of large 2D co-crystals.42 Briefly, with the SERCA inhibitory site in response to 100 μg of PLB and 600 μg of lipids (EYPC/EYPE/ EYPA in a physiological cues (elevated cytosolic calcium and/ weight ratio of 8:1:1) were solubilized in a chloroform- or phosphorylation). This interaction would also trifluoroethanol mixture, dried to a thin film under nitrogen explain how SERCA is able to influence the gas and lyophilized. Buffer (20 mM imidazole, pH 7.0, 63 oligomeric state of PLB. Based on their NMR 100 mM KCl, 0.02% (w/v) NaN3)anddetergent(C12E8)were added to solubilize the mixture, followed by the addition of structure of a PLB pentamer, Oxenoid and Chou μ suggested that individual PLB monomers could 500 g of detergent-solubilized, purified SERCA. It has been shown that this method of co-reconstitution yields complete initiate binding to SERCA without dissociation 50,78 41 recovery of SERCA transport activity and PLB inhibition. from the pentamer. We suggest a modification The final concentrations were adjusted to a 1:1:2 weight ratio of this hypothesis, where a PLB pentamer interacts of protein/lipid/detergents (final molar ratio of SERCA/ with the membrane domain of SERCA and serves PLB/lipids of approximately1:3.5:180). The detergent was as a reservoir for directed diffusion of monomers to removed by the slow addition of SM-2 Biobeads (25 mg of and from the inhibitory site (Fig. 8). This pathway wet beads) over 4 h. For the best results, crystallization was might be important in efficiently delivering mono- performed immediately on the co-reconstituted proteolipo- meric PLB to its binding site on SERCA, ensuring somes containing SERCA and PLB. Reconstituted proteoli- that the inhibited state is maintained following a posomes containing SERCA in the absence of PLB were cycle of calcium release in cardiac muscle. Specif- prepared simultaneously under identical conditions. ATPase ically, a site for the PLB pentamer on SERCA might activity of the proteoliposomes was measured by a coupled- enzyme assay over a range of calcium concentrations of position the monomer in a conformation that is 0.1 μM–10 μM.51,75 The apparent calcium affinity, K ,was compatible with formation of the inhibitory com- ca 41 determined by fitting the data to the Hill equation using plex, as suggested earlier. Furthermore, the Sigma Plot software (SPSS Inc., Chicago, IL). The functional resultant depolymerization of the PLB pentamer characterization of these mutants has been described by would leave behind a tetramer that might remain others17,55 as well as by our laboratory.51 associated with the pump. There is ample evidence For studies of the effect of PLB phosphorylation, wild type of intermediate oligomeric states for PLB50 and and K27A PLB were solubilized in detergent and phosphor- such intermediates could be poised to reacquire a ylated with the catalytic subunit of PKA; Sigma-Aldrich, St. PLB monomer once it dissociates from the inhibi- Louis, MO) before co-reconstitution with SERCA into tory complex with SERCA. Finally, phosphoryla- proteoliposomes. PKA was added to a concentration of 100 units/mg of detergent-solubilized PLB and the reaction tion has been shown to increase the oligomeric 16 was incubated at 30 °C for 3 h. It was concluded that this propensity of PLB and an interaction of these treatment resulted in complete phosphorylation of PLB, species with the pump could ensure a rapid return because the unphosphorylated protein could not be detected to the inhibited state upon dephosphorylation. either by MALDI-TOF mass spectrometry or by SDS-PAGE Otherwise, if allowed to diffuse away during a and western blotting (data not shown). period of β-adrenergic stimulation, there could be a substantial delay before SERCA randomly encoun- 2D crystallization ters a non-phosphorylated PLB molecule within the membrane plane. Co-reconstituted proteoliposomes were collected by cen- trifugation in crystallization buffer (20 mM imidazole, pH 7.4, 100 mM KCl, 35 mM MgCl2, 0.5 mM EGTA, 0.25 mM Materials and Methods μ 79 Na3VO4,30 M thapsigargin). The pellet was subjected to two freeze-thaw cycles, suspended with a micropipette, Octaethylene glycol monododecyl ether (C12E8) was followed by two more freeze-thaw cycles. Reconstituted obtained from Barnet Products (Englewood Cliff, NJ). samples were incubated at 4 °C for up to one week. Although SM-2 Biobeads were obtained from Bio-Rad (Hercules, crystallization occurred quickly, three to five days was CA). Egg yolk phosphatidylcholine (EYPC), egg yolk optimal for the highest frequency and quality of 2D crystals. phosphatidylethanolamine (EYPE) and egg yolk phos- phatidic acid (EYPA) were obtained from Avanti Polar Lipids (Alabaster, AL). All reagents used in the coupled Electron microscopy enzyme assay for measuring ATPase activity were of the highest purity available (Sigma-Aldrich, Oakville, ON Crystals were imaged in a Tecnai F20 electron micro- Canada).75 scope (FEI Company, Einhoven, Netherlands) in the 720 Co-crystals of SERCA and Phospholamban

Microscopy and Imaging Facility (University of Calgary) 3. Toyoshima, C. & Mizutani, T. (2004). Crystal structure or a JEOL 2200FS electron microscope (JEOL Ltd., Tokyo, of the calcium pump with a bound ATP analogue. Japan) in the Electron Microscopy Facility (National Nature, 430, 529–535. Institute for Nanotechnology, University of Alberta and 4. Toyoshima, C., Nomura, H. & Tsuda, T. (2004). National Research Council of Canada). Both microscopes Lumenal gating mechanism revealed in calcium were operated at 200 kV. A standard room temperature pump crystal structures with phosphate analogues. holder was used for negatively stained samples and a Nature, 432, 361–368. Gatan 626 cryoholder (Gatan Inc., Pleasanton, CA) was 5. Sorensen, T., Moller, J. & Nissen, P. (2004). Phosphoryl used for frozen-hydrated samples. Low-dose images were transfer and calcium ion occlusion in the calcium recorded either on film at a magnification of 50,000× pump. Science, 304, 1672–1675. (Tecnai F20) or image plates at a magnification of 35,800× 6. Olesen, C., Picard, M., Winther, A. M., Gyrup, C., (JEOL 2200FS). For film, the best images were digitized at Morth, J. P., Oxvig, C. et al. (2007). The structural basis 6.35 μm/pixel with a Nikon Super Coolscan 9000 followed of calcium transport by the calcium pump. Nature, by pixel averaging to achieve a final resolution of 2.54 Å/ 450, 1036–1042. pixel. The image plates were scanned at 15 μmpixel for a 7. Toyoshima, C., Norimatsu, Y., Iwasawa, S., Tsuda, T. final resolution of 4.44 Å/pixel. All data were recorded & Ogawa, H. (2007). How processing of aspartylpho- with defocus levels of 0.5–2 μm, with an emphasis on low- sphate is coupled to lumenal gating of the ion defocus images (0.5 μm and 1 μm) for the frozen-hydrated pathway in the calcium pump. Proc. Natl Acad. Sci. samples. USA, 104, 19831–19836. Projection maps were determined using the MRC image 8. 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