Structural basis for relief of phospholamban-mediated inhibition of the sarcoplasmic reticulum Ca2+-ATPase at saturating Ca2+ conditions

Sarcoplasmic reticulum Ca2+-ATPase (SERCA) is critical for cardiac Ca2+ transport. Reversal of phospholamban (PLB)-mediated SERCA inhibition by saturating Ca2+ conditions operates as a physiological rheostat to reactivate SERCA function in the absence of PLB phosphorylation. Here, we performed extensive atomistic molecular dynamics simulations to probe the structural mechanism of this process. Simulation of the inhibitory complex at superphysiological Ca2+ concentrations ([Ca2+] = 10 mm) revealed that Ca2+ ions interact primarily with SERCA and the lipid headgroups, but not with PLB's cytosolic domain or the cytosolic side of the SERCA–PLB interface. At this [Ca2+], a single Ca2+ ion was translocated from the cytosol to the transmembrane transport sites. We used this Ca2+-bound complex as an initial structure to simulate the effects of saturating Ca2+ at physiological conditions ([Ca2+]total ≈ 400 μm). At these conditions, ∼30% of the Ca2+-bound complexes exhibited structural features consistent with an inhibited state. However, in ∼70% of the Ca2+-bound complexes, Ca2+ moved to transport site I, recruited Glu771 and Asp800, and disrupted key inhibitory contacts involving the conserved PLB residue Asn34. Structural analysis showed that Ca2+ induces only local changes in interresidue inhibitory interactions, but does not induce repositioning or changes in PLB structural dynamics. Upon relief of SERCA inhibition, Ca2+ binding produced a site I configuration sufficient for subsequent SERCA activation. We propose that at saturating [Ca2+] and in the absence of PLB phosphorylation, binding of a single Ca2+ ion in the transport sites rapidly shifts the equilibrium toward a noninhibited SERCA–PLB complex.

The sarcoplasmic reticulum Ca 2ϩ -ATPase (SERCA) 2 uses the energy derived from hydrolysis of one ATP to transport two Ca 2ϩ ions from the cytosol into the lumen of the sarcoplasmic reticulum (SR) (1). In cardiac muscle cells, SERCA function is reversibly regulated by the transmembrane phospholamban (PLB). PLB binds SERCA in a 1:1 heterodimeric regulatory complex and inhibits SERCA activity (2,3). Phosphorylation of PLB then relieves SERCA inhibition (3) to increase the rate of cardiac muscle relaxation and to restore the SR Ca 2ϩ load necessary for muscle contraction in subsequent beats (4).
PLB inhibits SERCA by binding to a large pocket located in the transmembrane (TM) domain of the pump (5)(6)(7)(8)(9)(10). Spectroscopy studies have shown that in the bound complex, SERCA inhibition is tightly coupled to a structural transition between inhibitory and noninhibitory structural states of PLB (11)(12)(13)(14). More recently, X-ray crystallography studies showed that in its unphosphorylated form, PLB forms specific intermolecular interactions between conserved residue Asn 34 and residue Gly 801 of SERCA (15). Extensive studies by our group showed that these intermolecular interactions induce a substantial structural rearrangement of the transmembrane transport sites and stabilize a metal ion-free E1 intermediate of the pump protonated at residue Glu 771 , E1⅐H ϩ 771 (16,17). This SERCA intermediate serves as a kinetic trap that decreases SERCA's apparent affinity for calcium at low Ca 2ϩ concentrations and depresses the structural transitions necessary for Ca 2ϩ -dependent activation of SERCA (16,17).
In the absence of PLB phosphorylation, relief of SERCA inhibition occurs at saturating Ca 2ϩ concentrations (18,19). Two mechanisms have been proposed for the relief of SERCA inhibition at saturating Ca 2ϩ conditions: the dissociation model and the subunit model. The dissociation model proposes that PLB must physically separate from SERCA to relieve inhibition, whereas the subunit model hypothesizes that PLB acts as a functional subunit of SERCA, and inhibition is relieved by local structural rearrangements within the SERCA-PLB complex. Whereas cross-linking experiments have suggested that saturation Ca 2ϩ conditions induce PLB dissociation from SERCA, the findings from these studies are actually consistent with a structural rearrangement, and not dissociation, of the inhibitory complex (7,19). Furthermore, extensive spectroscopic experiments in live cells, ER membranes, and reconstituted vesicles unequivocally support the subunit model, as they provide direct detection of the SERCA-PLB interaction at high Ca 2ϩ concentrations (11, 20 -22).
These studies have shown that saturating Ca 2ϩ conditions do not induce dissociation of the SERCA-PLB complex, but the spatial and temporal mechanisms by which Ca 2ϩ reverses SERCA remain unknown. In this study, we designed a series of atomistic molecular simulations to determine the mechanisms for Ca 2ϩ -dependent relief of inhibitory interactions in the SERCA-PLB complex. First, we used a full-length structure of SERCA bound to the inhibitory structure of unphosphorylated PLB as a starting structure to obtain a structure of the SERCA-PLB complex at saturating Ca 2ϩ conditions. We then used the Ca 2ϩ -bound SERCA-PLB structural model generated through this computational approach to perform six independent 1-s molecular dynamics (MD) simulations of the complex at physiologically relevant conditions. This set of independent simulations was used to systematically identify the effects of saturating Ca 2ϩ conditions on the inhibitory contacts between SERCA and PLB in the absence of PLB phosphorylation.

Interaction of Ca 2؉ with the SERCA-PLB complex at saturating Ca 2؉ concentrations
Physiologically relevant saturating free Ca 2ϩ concentrations are in the low micromolar range (23), but these ion concentrations cannot be effectively modeled with our explicit MD simulations because the finite size of the systems would require less than one Ca 2ϩ ion/system. To overcome this limitation, we first performed a 0.5-s MD simulation of the SERCA-PLB complex at 10 mM CaCl 2 to mimic saturating Ca 2ϩ concentrations and to match the experimental conditions used previously in X-ray crystallography studies (24,25). At this Ca 2ϩ concentration, Ca 2ϩ ions interact with several acidic residues of SERCA exposed on both sides of the lipid bilayer, as well as with the lipid headgroups (Fig. 1). However, we found no indication that Ca 2ϩ interacts with the cytosolic domain of PLB or near the cytosolic side of the SERCA-PLB interface (Fig. 1).
Mapping of Ca 2ϩ -SERCA interactions revealed that Ca 2ϩ ions do not form interactions with functionally important regions of the pump, such as the phosphorylation site (Asp 351 ) or the K ϩ -binding site involved in SERCA dephosphorylation (Ala 711 , Ala 714 , Lys 712 , and Glu 732 ) (26) (Fig. 1). In other cases, Ca 2ϩ ions interact, albeit nonspecifically, with other functional sites in the cytosolic headpiece, such as the N␤5-␤6 loop (Asp 426 -Lys 436 ) (27) (Fig. 1). We found that Ca 2ϩ ions bind to SERCA near the cytosolic gate that leads to the transport sites ( Fig. 1). This site is located ϳ20 Å away from the SERCA-PLB interface and is not physically altered by PLB, and SERCAmetal ion interactions in this site are completely independent from PLB binding (15,16). On average, 2-3 Ca 2ϩ ions occupy this region of the protein, but only a single Ca 2ϩ ion binds to residues Asp 59 and Glu 309 at the entrance of the cytosolic pathway ( Fig. 2A). We found that in the submicrosecond timescale (t ϭ 0.34 s), Glu 309 translocates a single Ca 2ϩ ion from Asp 59 to Asp 800 (Fig. 2B). Ca 2ϩ translocation is facilitated by a change in the dihedral angle N-C␣-C␤-C␥ ( 1 ) of Glu 309 from values of ϩ165°and Ϫ165°to a 1 ϭ Ϫ65°. Upon translocation, Ca 2ϩ is stabilized in the transport sites by electrostatic interactions with residues Asp 800 and Glu 309 (Fig. 2C). This Ca 2ϩ -bound configuration of the complex is stable for the remainder of the simulation time, so we used it as starting structure for six independent MD simulations, identified as CAL1-CAL6. These MD simulations were used to determine the effects of saturating Ca 2ϩ conditions at physiologically relevant conditions (e.g. ϳ400 M total Ca 2ϩ and 100 mM K ϩ ).
This set of MD simulations revealed that Ca 2ϩ remains bound to the transport sites of SERCA and does not dissociate back to the cytosol at physiological conditions. We calculated the coordination number and coordination shell of Ca 2ϩ to characterize the interactions that stabilize a single Ca 2ϩ ion in the transport sites of SERCA. We define the coordination number of Ca 2ϩ as the number of oxygen atoms within 3.5 Å of the calcium ion. This distance is normally considered to be the maximum possible distance between ligand oxygen atoms and the calcium ion (28 -30). We found that in all trajectories, Ca 2ϩ interacts with the transport sites predominantly with a coordination number of 7, although Ca 2ϩ also exhibits a coordination number of 8 in a small percentage of the simulation time (Ͻ10%). These coordination numbers fall within the typical values estimated from Ca 2ϩ -protein (31, 32) and Ca 2ϩ -SERCA complexes (24,25).
In two trajectories, CAL1 and CAL3, the Ca 2ϩ ion interacts predominantly with seven coordinating oxygen atoms primarily in a pentagonal bipyramidal coordination geometry, in  Mechanism for Ca 2؉ binding to the transport sites at superphysiological Ca 2؉ concentrations. A, in the nanosecond timescale, a single Ca 2ϩ ion binds to SERCA residues Asn 59 and Glu 309 , located at the entrance of the cytosolic pathway leading to the transport sites. B, following Ca 2ϩ binding, rotation of the Glu 309 side chain facilitates translocation of Ca 2ϩ through the cytosolic pathway into the transport sites. C, upon translocation, the position of Ca 2ϩ in the transport sites is stabilized by electrostatic interactions with residues Asp 800 and Glu 309 for the remainder of the simulation time. In all panels, the TM helices are represented by gray ribbons, transport site residues are shown as sticks, and the Ca 2ϩ ion is represented as a yellow sphere.

Ca 2؉ -induced relief of SERCA inhibition by phospholamban
agreement with previous crystallographic studies of Ca 2ϩbound SERCA (25) (Fig. 3A). The seven coordinating ligands for Ca 2ϩ are the carboxylic oxygen atoms from residues Glu 309 and Asp 800 , the carbonyl moiety from residue Asn 796 , and between two and four water molecules (Fig. 3A). We found that in trajectories CAL2 and CAL4 -CAL6, Ca 2ϩ also coordinates to oxygen atoms within the transport site predominantly in a heptavalent pentagonal bipyramidal geometry. However, Glu 771 rapidly (t ϭ 25-250 ns) replaces Glu 309 in the first coordination shell of Ca 2ϩ (Fig. 3B). In this binding mode, Ca 2ϩ interacts with the oxygen atoms from transport site residues Glu 771 and Asp 800 , the carbonyl group from residue Asn 796 , and 2-3 water molecules (Fig. 3B).

Effect of saturating Ca 2؉ conditions on PLB-induced inhibitory interactions
Recent studies have shown that PLB residue Asn 34 , which is absolutely required for SERCA inhibition (34), forms specific hydrogen bond interactions with Gly 801 and Thr 805 in the TM domain of SERCA (15)(16)(17). These interactions induce alterations in the transport site geometry that prevent metal ion occlusion in the transport sites (16,17). The SERCA-Ca 2ϩ interactions shown in Fig. 3 suggest that saturating Ca 2ϩ concentrations alter SERCA-PLB inhibitory contacts at physiological conditions. Therefore, we measured intermolecular residue pair distances between Gly 801 and Asn 34 and between Thr 805 and Asn 34 . SERCA residues Glu 771 and Asp 800 play a key role in Ca 2ϩ occlusion in the transport sites (35), so we also measured the interresidue distance between Glu 771 and Asp 800 .
In the trajectories where Ca 2ϩ primarily binds to SERCA residues Glu 309 and Asp 800 (trajectories CAL1 and CAL3), PLB residue Asn 34 interacts directly with the backbone oxygen of Gly 801 and the side-chain hydroxyl group of Thr 805 (Fig. 4). In both cases, the intermolecular interactions Asn 34 -Gly 801 and Asn 34 -Thr 805 are present for most of the simulation time. Furthermore, we found that the carboxyl groups of transport site residues Glu 771 and Asp 800 in these trajectories are separated by a distance of at least 9 Å (Fig. 4). This spatial separation is characteristic of the inhibited SERCA-PLB complex in the absence of Ca 2ϩ (15,16). Therefore, the stability of the inhibitory interactions and the large spatial separation between Glu 771 and Asp 800 indicate that the structures populated in the trajectories CAL1 and CAL3 correspond to those of an inhibited Ca 2ϩ -bound state of the SERCA-PLB complex (15)(16)(17).
In four MD trajectories, CAL2, CAL4, CAL5, and CAL6, we found substantial changes in the distances between intermolecular residue pairs Asn 34 -Gly 801 and Asn 34 -Thr 805 . Here, the distance Asn 34 -Thr 805 increases by 1-2 Å (Fig. 4); however, the most significant change is the 3-Å increase in the distance between the side chain of Asn 34 and the backbone oxygen of Gly 801 (Fig. 4). Most importantly, we found that the spatial separation Asn 34 of PLB and Gly 801 of SERCA in these trajectories occurs concomitantly with a 3-4-Å decrease in the distance between transport site residues Glu 771 and Asp 800 (Fig. 4). These changes in interresidue distances are also accompanied by a shift in the dihedral angle C␣-C␤-C␥-N␦ ( 2 ) of Asn 34 from two narrow distributions at 2 ϭ ϩ180°and 2 ϭ Ϫ180°to a single broad distribution with a mean around Ϫ20° (Fig. 5). This structural change is mostly characterized by a transition from an extended side-chain conformation to a self-contact (36) involving side-chain nitrogen and backbone oxygen atoms of Asn 34 (Fig. 5). These findings indicate that in trajectories CAL2, CAL4, CAL5, and CAL6, the side chain of PLB residue Asn 34 becomes more mobile and no longer establishes inhibitory contacts with SERCA.
These observations provide evidence that is consistent with relief of SERCA-PLB inhibition at saturating Ca 2ϩ conditions. However, this phenomenon is not consistently observed in all six trajectories, which suggests that this disruption of inhibitory Here, Ca 2ϩ interacts with Glu 309 and Asp 800 in either monodentate or bidentate coordination geometries; the carboxamide moiety of Asn 796 and water molecules are also found in the first coordination shell of Ca 2ϩ . B, structures of the most populated Ca 2ϩ coordination geometries found in the trajectories CAL2, CAL4, CAL5, and CAL6. Here, the carboxylic groups of Glu 771 and Asp 800 act as monodentate (e.g. structures 1 and 2) or bidentate ligands (e.g. structure 3) to bind Ca 2ϩ in the transport sites. The coordination geometry of Ca 2ϩ in these trajectories also includes Asn 796 and 2-3 water molecules. In all panels, transport site residues and water molecules are shown as sticks, oxygen atoms in the first coordinating shell of Ca 2ϩ are shown as red spheres, and the Ca 2ϩ ion is represented as a yellow sphere. Distances between key inhibitory contacts involving conserved PLB residue Asn 34 and SERCA Gly 801 were calculated using atoms N ␦ of Asn 34 and the backbone oxygen of Gly 801 . The distance between Thr 805 and Asn 34 was calculated between the O ␥ of Thr 805 and N ␦ of Asn 34 . Distances between Glu 771 and Asp 800 , which occlude Ca 2ϩ in site I, were calculated using atoms C ␦ and C ␥ , respectively. The dashed lines indicate the interresidue distances in the initial structure-inhibited complex reported in a previous study by our group (16).

Ca 2؉ -induced relief of SERCA inhibition by phospholamban
contacts probably occurs in equilibrium and in a Ca 2ϩ -independent manner. To test this hypothesis, we performed six 1-s MD simulations of the SERCA-PLB complex in the absence of Ca 2ϩ . We found that whereas intermolecular distance Asn 34 -Thr 805 is variable among independent MD trajectories, Asn 34 of PLB consistently remains spatially close to SERCA residue Gly 801 throughout the entire simulation time in all trajectories (Fig. 6). We also found that carboxylic groups of transport site residues Glu 771 and Asp 800 is are separated by a distance larger than 9 Å in all Ca 2ϩ -free trajectories (Fig. 6); such distance is longer than the 6-Å separation required for Ca 2ϩ occlusion in this site (24,25). Therefore, these findings indicate that relief of SERCA-PLB inhibitory contacts does not occur spontaneously in the absence of Ca 2ϩ and that Ca 2ϩ binding to SERCA at saturating Ca 2ϩ conditions disrupts key SERCA-PLB inhibitory contacts at physiological conditions.

Effects of saturating Ca 2؉ conditions on the structural dynamics of PLB
Our results demonstrate that Ca 2ϩ binding to the transport sites of SERCA at saturating Ca 2ϩ conditions generally disrupt key inhibitory interactions between SERCA and PLB. However, it is not clear whether disruption of the inhibitory interactions is linked to (i) changes in the structural dynamics of PLB, (ii) a reorganization of the SERCA-PLB interface, or (iii) local changes involving interresidue interactions along the interface. Therefore, we performed extensive measurements of structural parameters to determine the changes in the structural dynamics of PLB in response to saturating Ca 2ϩ conditions.
In the both inhibited and noninhibited Ca 2ϩ -bound complexes, the cytosolic and TM helices that contain the regulatory phosphorylation and inhibitory domains populate an ␣-helical structure for Ͼ95% of the time. Average interhelical angles between the cytosolic (Val 4 -Thr 17 ) and TM (residues Arg 25 -Leu 52 ) helices of PLB fluctuate between 52 and 77° (Table 1), which corresponds to a T-shaped architecture of PLB (Fig. 7A). We found that the calculated interhelical angles of PLB in both inhibited and noninhibited complexes are within the range of those determined experimentally for the unphosphorylated PLB monomer in solution (37,38). These findings are in agreement with previous spectroscopic studies (11,39) and demonstrate that saturating Ca 2ϩ conditions do not induce order-todisorder transitions associated with PLB phosphorylation (12, 39 -43).
We measured time-dependent root mean square deviation (RMSD) to determine the extent to which the position of the TM domain of PLB changes in the trajectories of the Ca 2ϩbound complexes. RMSD plots revealed that the position of PLB in the binding groove in all six trajectories does not deviate substantially (e.g. RMSD Ͻ 2.5 Å) from that determined by X-ray crystallography (Fig. 7B). In addition, root mean square fluctuation (RMSF) calculations using the main-chain C␣ atoms show that the cytosolic helix of PLB is highly mobile in solution (Fig. 7C). This behavior corresponds to the inherent diffusion of the helical domain through the viscous bilayer surface, and it is uncorrelated with the presence or absence of inhibitory contacts. The RMSF values of the TM domain residues in all MD trajectories are substantially smaller than those calculated for the cytosolic helix; this indicates that the TM domain of PLB has very low mobility in the microsecond time scale. The RMSF values calculated for the TM domain of PLB, and particularly those around the residue Asn 34 , are virtually identical both in the presence and absence of intermolecular inhibitory contacts (Fig. 7C).
We also calculated changes in the tilt angle of the TM helix of PLB to complement RMSD and RMSF measurements. We measured the relative tilt angle of the TM helix using the crystal structure of SERCA-PLB as a reference (Table 1). We found that the TM helix exhibits on average a 3.6°increase in the tilt angle relative to the position of PLB in the crystal structure of the complex (Table 1). We found no correlation between the loss of inhibitory contacts and the small change in tilt angle, which indicates that saturating Ca 2ϩ conditions do not have an effect on the position of PLB in the complex. Hence, it is likely that the small changes in tilt angle are inherent to the PLB  Relief of inhibitory contacts is not accompanied by substantial changes in the structure of PLB in the complex, so disruption of SERCA-PLB inhibitory contacts must occur locally at the interface of the complex. To test this hypothesis, we measured the fraction of native inhibitory contacts, Q inh , between SERCA and PLB residues Leu 31 , Asn 34 , Phe 35 , and Ile 38 ; these residues, located near the cytosolic side of the complex, play important roles in inhibition of SERCA (45). Analysis of the Q inh values showed that in the complexes with intact inhibitory contacts (CAL1 and CAL3), there is a high retention of native inhibitory contacts (Q inh Ͼ 0.8) between key PLB residues and SERCA ( Table 1). As anticipated, there is a substantial decrease in native inhibitory contacts (Q inh ϭ 0.52-0.62) for PLB residue Asn 34 in the trajectories where inhibitory contacts are disrupted (CAL2, CAL4, CAL5, and CAL6; Table 1). However, we found that a loss in native inhibitory contacts also occurs, albeit more moderately, at PLB positions Leu 31 , Phe 35 , and Ile 38 (Table 1). This indicates that saturating Ca 2ϩ conditions primarily affect local intermolecular interactions involving the side chain of PLB residue Asn 34 , but also affect intermolecular interactions involving PLB residues within the inhibitory site at the SERCA-PLB interface.

Structure of the transport sites upon Ca 2؉ -induced disruption of SERCA-PLB inhibitory interactions
We determined whether the structural changes following disruption of inhibitory SERCA-PLB interactions correspond to those associated with the formation of competent transport sites. To this aim, we measured the RMSD values for residues in sites I and II between the MD trajectories and the crystal structure of SERCA bound to two Ca 2ϩ ions (E1⅐2Ca 2ϩ , PDB entry 1SU4). We also performed distance measurements to determine whether the location of the Ca 2ϩ in the MD trajectories corresponds to that determined by X-ray crystallography (25).
Visualization of the transport sites in the trajectories CAL1 and CAL3, where the inhibitory SERCA-PLB interactions are intact, show that sites I and II are collapsed (Fig. 8). Calculated RMSD values for sites I and II (RMSD Ͼ2.7 Å) indicate that there is a poor overlap between these trajectories and the crystal structure of E1⅐2Ca 2ϩ (Table 2). In these trajectories, the position of Ca 2ϩ does not overlap with any of the two sites resolved by X-ray crystallography (Fig. 8). Instead, the Ca 2ϩ ion binds to a location that is distant from sites I (r Ϸ 5.5 Å) and site II (r Ϸ 3.5 Å). These measurements indicate that in the presence of inhibitory SERCA-PLB contacts, Ca 2ϩ does not bind to either site I or II, and the residues in the transport sites adopt a noncompetent structure.

Ca 2؉ -induced relief of SERCA inhibition by phospholamban
In the MD simulations where the inhibitory SERCA-PLB are disrupted (trajectories CAL2, CAL4, CAL5, and CAL6), the structure of transport site II also deviates substantially from that in the crystal structure of E1⅐2Ca 2ϩ (Fig. 8 and Table 2). However, we found that Ca 2ϩ ion binds in a reproducible manner to a location that partially overlaps that of site I (r Ϸ 2 Å) determined by X-ray crystallography ( Fig. 8 and Table 2). When a single Ca 2ϩ ion binds to this site, residues Glu 771 , Thr 799 , Asp 800 , and Glu 908 of site I adopt a structural arrangement that is similar to that found in the crystal structure of the PLB-free E1⅐2Ca 2ϩ state of SERCA (RMSD Ͻ2.2 Å; Fig. 8). The structural rearrangements in the transport sites that follow Ca 2ϩ -induced relief of SERCA inhibition are reproducible in trajectories CAL2 and CAL4 -CAL6 (Fig. 8) and correspond to the formation of a competent site I.

Discussion
We present a mechanistic study of the SERCA-PLB regulatory interactions at saturating Ca 2ϩ conditions, thus providing quantitative insight into fundamental processes of activation of Ca 2ϩ transport in the heart. We show that in a solution containing 10 mM Ca 2ϩ , calcium ions interact primarily with both cytosolic and luminal sides of SERCA and the lipid headgroups.
Here, we show for the first time (to our knowledge) that at superphysiological Ca 2ϩ conditions, Ca 2ϩ ions interact with the luminal C-terminal region of PLB, but not with the cytosolic domain of PLB or the cytosolic side of the SERCA-PLB interface. This indicates that Ca 2ϩ does not compete with PLB at the interface of the complex and does not have a direct effect on the structural dynamics and stability of unphosphorylated PLB. Previous FRET spectroscopy experiments support our data and show that saturating Ca 2ϩ conditions alter neither the structural dynamics of unphosphorylated PLB nor the stability of the SERCA-PLB heterodimer (11).
At [Ca 2ϩ ] ϭ 10 mM, Ca 2ϩ ions interact with SERCA at the entrance of the pathway that connects the cytosol with the transport sites, in agreement with previous studies by our group showing that PLB does not block metal ion binding to this region of SERCA (16,17). Recognition of Ca 2ϩ by SERCA is facilitated primarily by residues Asp 59 and Glu 309 , in agreement with mutagenesis studies of the pump (46). We found that Glu 309 translocates a single Ca 2ϩ ion from Asp 59 to Asp 800 , a critical residue in the transport sites that coordinates Ca 2ϩ at sites I and II (25). This mechanism for Ca 2ϩ translocation is in qualitative agreement with Brownian dynamics studies showing that fast Ca 2ϩ binding to the transport sites is primarily guided by Glu 309 (47).
In the absence of adequate charge neutralization of the transmembrane transport sites, SERCA denaturalization occurs very rapidly even within the native membrane at physiological pH (48,49). Previous studies have shown that this electric charge can be compensated in the absence of Ca 2ϩ by transport site protonation (16,17) or by binding of metal ion K ϩ (50), Na ϩ (51-53), or Mg 2ϩ (54,55). This suggests that superphysiological concentrations of Ca 2ϩ used in this study simply satisfy transport site charge neutralization and that the Ca 2ϩ -bound state of the SERCA-PLB complex might not represent a functional state in the cell. However, only a single Ca 2ϩ ion occupies the transport sites at a time despite the superphysiological Ca 2ϩ concentrations used in this study. This finding is consistent with previous studies showing that the Ca 2ϩ binding to SERCA occurs in a sequential manner (56 -58) and indicates that the Ca 2ϩ -bound SERCA-PLB structure represents a functional state of the complex at saturating Ca 2ϩ conditions. In the absence of other Ca 2ϩ ions, a single Ca 2ϩ bound to the SERCA-PLB complex produces a total [Ca 2ϩ ] of ϳ400 M. This value falls in the middle of previous estimates at elevated cytosolic Ca 2ϩ in the cardiomyocyte (59 -61), so we used the Ca 2ϩbound SERCA-PLB complex as a starting structure to probe the structural mechanism for relief of SERCA inhibition by PLB.
In 30% of the MD trajectories, Ca 2ϩ binds to Glu 309 and Asp 800 in an orientation that is similar to that initially found at the end of the 0.5-s MD trajectory at [Ca 2ϩ ] ϭ 10 mM. In this configuration, the inhibitory contacts remain intact in the microsecond time scale, and transport sites I and II lack the competent structural organization that is distinctive of the Ca 2ϩ -bound state of SERCA (25,53,62). This structural arrangement corresponds to a Ca 2ϩ -bound, inhibited SERCA-PLB complex. Previous studies have shown that PLB binding decreases SERCA's apparent affinity for Ca 2ϩ only by 2-3-fold Representative structures extracted from each MD simulation illustrate the location of Ca 2ϩ (yellow sphere) and the structural arrangement of the residues that occlude Ca 2ϩ in the transport sites (orange sticks). For comparison, each MD structure was superposed on the crystal structure of the Ca 2ϩ -bound SERCA, E1⅐2Ca 2ϩ (25), to show the structure and location of the competent transport sites I and II; the side chains and Ca 2ϩ ions resolved in the crystal structure are shown as blue sticks and cyan spheres, respectively. Based on the structural characteristics of each configuration, the structures of the transport sites are labeled as inhibited or not inhibited.  (25)) as reference. c Distances relative to the position of Ca 2ϩ in sites I and II determined by x-ray crystallography (25).

Ca 2؉ -induced relief of SERCA inhibition by phospholamban
in the micromolar range (63), whereas others have suggested that PLB suppresses Ca 2ϩ binding to SERCA (64). Our simulations help reconcile these conflicting studies because they show that PLB-induced changes in the transport sites delay Ca 2ϩ binding to either sites I or II, thus altering the apparent Ca 2ϩ affinity of SERCA (34). SERCA-PLB inhibitory interactions are disrupted in 70% of the MD trajectories of the Ca 2ϩ -bound complex. In these cases, the initially bound Ca 2ϩ moves to site I and recruits the carboxylic groups of transport site residues Glu 771 and Asp 800 . These Ca 2ϩ -induced structural changes occur concomitantly with a loss in the intermolecular interaction between the side chain of PLB residue Asn 34 and the backbone oxygen of SERCA residue Gly 801 . Our data indicates that this Ca 2ϩ -dependent relief inhibitory contacts does not result from a large structural rearrangements in the SERCA-PLB interface (22) or changes in the native structural dynamics of PLB in the complex. Instead, PLB remains bound to SERCA, but PLB residue Asn 34 becomes dynamically more disordered and is unable to establish inhibitory contacts with SERCA. FRET spectroscopy experiments support these findings and show that Ca 2ϩ acts upon SERCA-PLB complex exclusively at the TM domain level and that unlike PLB phosphorylation, Ca 2ϩ does not induce changes in the structural dynamics of PLB (11). Whereas these structural changes have not been observed directly by spectroscopy, the Ca 2ϩ -induced repositioning and mobility of PLB residue Asn 34 observed in our simulations have been seen in X-ray crystallography studies of the complex at [Ca 2ϩ ] ϭ 1 mM. 3 What are the specific interactions between Ca 2ϩ and the transport sites that induce relief of inhibition? The crystal structure of the complex between sarcolipin, a PLB analog, and SERCA revealed a single Mg 2ϩ ion bound to transport site residues Glu 771 and Asp 800 (55). In this structure, the intermolecular inhibitory interactions are partially altered, which suggests that binding of divalent metal ions in the transport sites is sufficient to reverse SERCA inhibition. However, extensive studies by our group have demonstrated that in the inhibitory complex, Mg 2ϩ does not simultaneously interact with Asp 800 and Glu 771 (17). Instead, Mg 2ϩ adopts a rigid octahedral coordination geometry that has a preference for binding water molecules as opposed to bulky protein side chain dipoles (17). In addition, the ionic radius of Mg 2ϩ is smaller than that of Ca 2ϩ (65), so adding side chain dipoles to the coordination shell is thermodynamically more favorable for Ca 2ϩ than for Mg 2ϩ , so Ca 2ϩ can produce drier, bulkier coordination complexes (66,67). This explains why Ca 2ϩ , but not Mg 2ϩ , recruits both Glu 771 and Asp 800 in the transport sites (17,54). Owing to these distinctive properties of Ca 2ϩ , the tug of war between the attraction of Glu 771 and Asp 800 for Ca 2ϩ drag the Gly 801 backbone along with them as they move in toward Ca 2ϩ . These Ca 2ϩ -induced changes destabilize the interaction between Gly 801 and PLB residue Asn 34 and induce relief of SERCA inhibition by PLB. It is our postulate that Glu 771 and Asp 800 to a large degree define the range of coordination spheres that help preserve or disrupt the inhibitory contacts in the SERCA-PLB complex.
Finally, we asked whether the Ca 2ϩ -induced structural changes detected in our simulations produce an intermediate state in the pathway toward SERCA activation. First, we found that upon relief of inhibitory contacts, the side chain of Glu 309 populates a geometry in which the carboxylic group points toward the cytosol. We propose that this orientation of Glu 309 is essential for binding and gating of a second Ca 2ϩ ion in the transport site II (68). Second, we found that relief of inhibitory SERCA-PLB interactions occurs only when a single Ca 2ϩ binds near transport site I and in agreement with mutagenesis studies showing that binding of a single Ca 2ϩ in the transport site I is sufficient to reverse SERCA inhibition by PLB (19). The Ca 2ϩinduced structural rearrangements we detected in the simulations correspond to those associated with the formation of a competent transport site I and a vacant site II. This transport site preorganization facilitates binding of a second Ca 2ϩ ion and subsequent Ca 2ϩ -induced activation of the pump (25,50,53,62).
In summary, we demonstrate that at saturating Ca 2ϩ concentrations, binding of a single Ca 2ϩ ion shifts the equilibrium toward a noninhibited structure of the SERCA-PLB complex.
Our findings indicate that Ca 2ϩ -induced reversal of SERCA inhibition depends solely on the ability of Ca 2ϩ to diffuse into the transport sites and that the ability of Ca 2ϩ to enter the transport sites is not influenced by PLB. Our findings also indicate that reversal of SERCA-PLB inhibition at saturating Ca 2ϩ conditions is uncoupled from other regulatory mechanisms, such as the order-to-disorder structural transitions of PLB (12, 39 -43). Therefore, the lack of a regulatory mechanism would explain the inability of saturating [Ca 2ϩ ] to effectively reverse impaired SERCA-mediated Ca 2ϩ transport (4,70,71) and maladaptations (72) characteristic of chronic heart failure.

Setting up SERCA-PLB at superphysiological concentrations of Ca 2؉
We used an atomic model of the full-length PLB bound to SERCA generated previously by our group (16) to simulate the inhibited SERCA-PLB complex at superphysiological Ca 2ϩ conditions. We modeled transport site residues Glu 309 , Glu 771 , and Asp 800 as unprotonated and residue Glu 908 as protonated. In addition, we adjusted the pK a of other ionizable residues to a pH value of ϳ7.2 using PROPKA version 3.1 (73,74). The complex was inserted in a pre-equilibrated 120 ϫ 120-Å bilayer of palmitoyl-2-oleoyl-sn-glycerol-phosphatidylcholine lipids. We used the first-layer phospholipids that surround SERCA in the E1 state (75) as a reference to insert the complex in the lipid bilayer. This initial system was solvated using TIP3P water molecules with a minimum margin of 15 Å between the protein and the edges of the periodic box in the z axis. Ca 2ϩ and Cl Ϫ ions were added to produce a CaCl 2 concentration of ϳ10 mM required to match the experimental conditions previously used to obtain crystal structures of Ca 2ϩ -bound SERCA (24,25).

Setting up the SERCA-PLB complex at saturating Ca 2؉ conditions
We used the structure of the complex bound to a single Ca 2ϩ ion obtained at superphysiological [Ca 2ϩ ] as a starting structure to simulate the SERCA-PLB complex at saturating Ca 2ϩ conditions. We found that a single Ca 2ϩ ion bound to the transport sites of SERCA corresponds to a total Ca 2ϩ concentration of ϳ400 M. This total Ca 2ϩ concentration is much higher than that estimated at rest (76) and is also in good agreement with previous estimates at elevated cytosolic Ca 2ϩ (59 -61). The SERCA-PLB-Ca 2ϩ -lipid complex was solvated using TIP3P water molecules. K ϩ and Cl Ϫ ions were added to neutralize the system and to produce a KCl concentration of ϳ100 mM.

Setting up the SERCA-PLB complex at free Ca 2؉ conditions
We used an atomic model of the full-length SERCA-PLB structure (16) to simulate the inhibited complex at free Ca 2ϩ conditions. On the basis of our previous studies (16), we modeled transport site residues Glu 309 and Asp 800 as unprotonated and residues Glu 771 and Glu 908 as protonated. The lipidwater-protein complex was prepared using the same protocol and KCl concentrations used for the complex at saturating Ca 2ϩ conditions.

Molecular dynamics simulations
MD simulations of all systems were performed by using the program NAMD version 2.12 (77), with periodic boundary conditions (78), particle mesh Ewald (79,80), a nonbonded cutoff of 9 Å, and a 2-fs time step. CHARMM36 force field topologies and parameters were used for the proteins (81), lipid (69), water, Ca 2ϩ , K ϩ , and Cl Ϫ . The NPT ensemble was maintained with a Langevin thermostat (310 K) and an anisotropic Langevin piston barostat (1 atm). Fully solvated systems were first subjected to energy minimization and warmup for 200 ps. This procedure was followed by 10 ns of equilibration with backbone atoms harmonically restrained using a force constant of 10 kcal mol Ϫ1 Å Ϫ2 . We performed one 0.5-s MD simulation of SERCA-PLB at 10 mM Ca 2ϩ and 12 independent 1-s MD simulations: six of Ca 2ϩ -bound SERCA-PLB and six of SERCA-PLB in the absence of Ca 2ϩ .

Structural analysis and visualization
VMD (33) was used for analysis, visualization, and rendering of the structures. To visualize the Ca 2ϩ -protein and Ca 2ϩlipid interactions, we created a map of the weighted mass density of Ca 2ϩ using a grid resolution of 1 Å and a cutoff distance of 3.5 Å between Ca 2ϩ and the protein/lipid atoms. This is achieved by replacing each atom in the selection with a normalized Gaussian distribution of width equal to the atomic radius. The distributions are then additively distributed on a grid. The final map is calculated by computing the mass density of Ca 2ϩ for each step in the trajectory and averaged over the entire simulation time.
We calculated the fraction of native inhibitory contacts (Q inh ) between PLB residues Leu 31 , Asn 34 , Phe 35 , and Ile 38 and SERCA to measure the effect of calcium binding on the stability of the SERCA-PLB interface. Q inh is defined by a list of native contact pairs (i,j) in the crystal structure of the complex. All pairs of heavy atoms i and j belonging to residues Xi and Xj are in contact if the distance between i and j is Ͻ7 Å. Q inh is expressed as a number between 1 and 0, and it is calculated as the total number of native contacts for a given time frame divided by the total number of contacts in the crystal structure of the complex (PDB code 4KYT (15)).