Structures of the heart specific SERCA2a Ca2+-ATPase.

The sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) performs active reuptake of cytoplasmic Ca2+ and is a major regulator of cardiac muscle contractility. Dysfunction or dysregulation of SERCA2a is associated with heart failure, while restoring its function is considered as a therapeutic strategy to restore cardiac performance. However, its structure has not yet been determined. Based on native, active protein purified from pig ventricular muscle, we present the first crystal structures of SERCA2a, determined in the CPA-stabilized E2-AlF4- form (3.3 Å) and the Ca2+-occluded [Ca2]E1-AMPPCP form (4.0 Å). The structures are similar to the skeletal muscle isoform SERCA1a pointing to a conserved mechanism. We seek to explain the kinetic differences between SERCA1a and SERCA2a. We find that several isoform-specific residues are acceptor sites for post-translational modifications. In addition, molecular dynamics simulations predict that isoform-specific residues support distinct intramolecular interactions in SERCA2a and SERCA1a. Our experimental observations further indicate that isoform-specific intramolecular interactions are functionally relevant, and may explain the kinetic differences between SERCA2a and SERCA1a.

An N-terminal Ca2+-binding motif regulates the secretory pathway Ca2+/Mn2+-transport ATPase SPCA1.

The Ca2+/Mn2+ transport ATPases 1a and 2 (SPCA1a/2) are closely related to the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and are implicated in breast cancer and Hailey-Hailey skin disease. Here, we purified the human SPCA1a/2 isoforms from a yeast recombinant expression system and compared their biochemical properties after reconstitution. We observed that the purified SPCA1a displays a lower Ca2+ affinity and slightly lower Mn2+ affinity than SPCA2. Remarkably, the turnover rates of SPCA1a in the presence of Mn2+ and SPCA2 incubated with Ca2+ and Mn2+ were comparable, whereas the turnover rate of SPCA1a in Ca2+ was 2-fold higher. Moreover, we noted an unusual biphasic activation curve for the SPCA1a ATPase and autophosphorylation activity, not observed with SPCA2. We also found that the biphasic pattern and low apparent ion affinity of SPCA1a critically depends on ATP concentration. We further show that the specific properties of SPCA1a at least partially depend on an N-terminal EF-hand-like motif, which is present only in the SPCA1a isoform and absent in SPCA2. This motif binds Ca2+, and its mutation lowered the Ca2+ turnover rate relative to that of Mn2+, increased substrate affinity, and reduced the level of biphasic activation of SPCA1a. A biochemical analysis indicated that Ca2+ binding to the N-terminal EF-hand-like motif promotes the activity of SPCA1a by facilitating autophosphorylation. We propose that this regulation may be physiologically relevant in cells with a high Ca2+ load, such as mammary gland cells during lactation, or in cells with a low ATP content, such as keratinocytes.

Structure/activity relationship of thapsigargin inhibition on the purified Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a).

The Golgi/secretory pathway Ca2+/Mn2+-transport ATPase (SPCA1a) is implicated in breast cancer and Hailey-Hailey disease. Here, we purified recombinant human SPCA1a from Saccharomyces cerevisiae and measured Ca2+-dependent ATPase activity following reconstitution in proteoliposomes. The purified SPCA1a displays a higher apparent Ca2+ affinity and a lower maximal turnover rate than the purified sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1a). The lipids cholesteryl hemisuccinate, linoleamide/oleamide, and phosphatidylethanolamine inhibit and phosphatidic acid and sphingomyelin enhance SPCA1a activity. Moreover, SPCA1a is blocked by micromolar concentrations of the commonly used SERCA1a inhibitors thapsigargin (Tg), cyclopiazonic acid, and 2,5-di-tert-butylhydroquinone. Because tissue-specific targeting of SERCA2b by Tg analogues is considered for prostate cancer therapy, the inhibition of SPCA1a by Tg might represent an off-target risk. We assessed the structure-activity relationship (SAR) of Tg for SPCA1a by in silico modeling, site-directed mutagenesis, and measuring the potency of a series of Tg analogues. These indicate that Tg and the analogues are bound via the Tg scaffold but with lower affinity to the same homologous cavity as on the membrane surface of SERCA1a. The lower Tg affinity may depend on a more flexible binding cavity in SPCA1a, with low contributions of the Tg O-3, O-8, and O-10 chains to the binding energy. Conversely, the protein interaction of the Tg O-2 side chain with SPCA1a appears comparable with that of SERCA1a. These differences define a SAR of Tg for SPCA1a distinct from that of SERCA1a, indicating that Tg analogues with a higher specificity for SPCA1a can probably be developed.

Distinct roles of the C-terminal 11th transmembrane helix and luminal extension in the partial reactions determining the high Ca2+ affinity of sarco(endo)plasmic reticulum Ca2+-ATPase isoform 2b (SERCA2b).

The molecular mechanism underlying the characteristic high apparent Ca(2+) affinity of SERCA2b relative to SERCA1a and SERCA2a isoforms was studied. The C-terminal tail of SERCA2b consists of an 11th transmembrane helix (TM11) with an associated 11-amino acid luminal extension (LE). The effects of each of these parts and their interactions with the SERCA environment were examined by transient kinetic analysis of the partial reaction steps in the Ca(2+) transport cycle in mutant and chimeric Ca(2+)-ATPase constructs. Manipulations to the LE of SERCA2b markedly increased the rate of Ca(2+) dissociation from Ca(2)E1. Addition of the SERCA2b tail to SERCA1a slowed Ca(2+) dissociation, but only when the luminal L7/8 loop of SERCA1 was simultaneously replaced with that of SERCA2, thus suggesting that the LE interacts with L7/8 in Ca(2)E1. The interaction of LE with L7/8 is also important for the low rate of the Ca(2)E1P → E2P conformational transition. These findings can be rationalized in terms of stabilization of the Ca(2)E1 and Ca(2)E1P forms by docking of the LE near L7/8. By contrast, low rates of E2P dephosphorylation and E2 → E1 transition in SERCA2b depend critically on TM11, particularly in a SERCA2 environment, but do not at all depend on the LE or L7/8. This indicates that interaction of TM11 with SERCA2-specific sequence element(s) elsewhere in the structure is critical in the Ca(2+)-free E2/E2P states. Collectively these properties ensure a higher Ca(2+) affinity of SERCA2b relative to other SERCA isoforms, not only on the cytosolic side, but also on the luminal side.

Thapsigargin affinity purification of intracellular P(2A)-type Ca(2+) ATPases.

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca(2+) ATPase (SERCA2b) and secretory-pathway Ca(2+) ATPase (SPCA1a) belong both to the P(2A)-type ATPase subgroup of Ca(2+) transporters and play a crucial role in the Ca(2+) homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca(2+) ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump.

Sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) Ca(2+) transporters pump cytosolic Ca(2+) into the endoplasmic reticulum, maintaining a Ca(2+) gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca(2+) ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca(2+) affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure-function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca(2+) affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca(2+)-bound E1 conformation and alters Ca(2+)-transport kinetics, which provides the rationale for the higher apparent Ca(2+) affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the alpha- and beta-subunits of Na(+),K(+)-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca(2+) affinity of malfunctioning SERCA2a in the failing heart to improve contractility.

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+.

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.e., a twofold higher apparent Ca(2+) affinity, but twofold lower maximal turnover rate) can explain these compensatory changes, we simulated Ca(2+) dynamics in mouse ventricular myocytes. The model shows that the relative Ca(2+) transport capacity of SERCA2a and SERCA2b depends on the SERCA concentration. The simulations point to a dominant effect of SERCA2b's higher Ca(2+) affinity over its lower maximal turnover rate. The results suggest that increased systolic and decreased diastolic Ca(2+) levels in unstimulated conditions could contribute to the downregulation of SERCA in SERCA2(b/b) mice. In stress conditions, Ca(2+) handling is less efficient by SERCA2b than by SERCA2a, which might contribute to the observed hypertrophy in SERCA2(b/b) mice. Altogether, SERCA2a might be a better compromise between performance in basal conditions and performance during ß-adrenergic stress.

Time-Dependent Protein Thermostability Assay.

Membrane protein purification often yields rather unstable proteins impeding functional and structural protein characterization. Low protein stability also leads to low purification yields as a result of protein degradation, aggregation, precipitation, and folding instability. It is often required to optimize buffer conditions through numerous iterations of trial and error to improve the homogeneity, stability, and solubility of the protein sample demanding high amounts of purified protein. Therefore we have set up a fast, simple, and high-throughput time-dependent thermostability-based assay at low protein cost to identify protein stabilizing factors to facilitate the handling and characterization of membrane proteins by subsequent structural and functional studies.

High-throughput measurement of the Ca2+-dependent ATPase activity in COS microsomes.

We provide a detailed procedure to determine the Ca(2+)-dependent ATPase activity in COS or HEK293 cells overexpressing a Ca(2+) pump. The ATPase activity is determined by the Baginsky method, which allows measurement of the steady-state production of inorganic phosphate (Pi). We have adapted this widely applied method into a sensitive, fast, and semi-high-throughput protocol suitable for use in a 96-well plate format.

Measuring Ca2+-dependent Ca2+-uptake activity in the mouse heart.

The apparent Ca(2+) affinity of the isoforms of the sarco/endoplasmic reticulum Ca(2+) ATPase SERCA2 is controlled primarily by two proteins, phospholamban (PLB) and sarcolipin (SLN). The rate of ATP-driven Ca(2+) uptake into sarcoplasmic reticulum (SR)-derived vesicles can be monitored by a technique in which the net uptake of (45)Ca(2+) in the form of an intravesicular calcium oxalate precipitate is recorded. Here, we present details of a modification of such a protocol for determining the apparent Ca(2+) affinity of the Ca(2+) pump, and its control by various regulators, in crude homogenates of mouse heart.

Measuring Ca2+ pump activity in overexpression systems and cardiac muscle preparations.

Sarco-/endoplasmic reticulum (SR/ER) Ca(2+) pumps (SERCAs) build up vital Ca(2+) gradients across the intracellular SR/ER membrane, helping to control cell function, proliferation, growth, differentiation, and death. We describe two techniques to measure the SERCA activity either in mammalian culture cells overexpressing SERCAs or in muscle tissue containing high levels of endogenous SERCAs. As Ca(2+) transport is tightly coupled to ATP hydrolysis, it is possible to determine the rate of Ca(2+)-dependent ATP hydrolysis and use it as a measure for SERCA activity or, in a second approach, to quantify ATP-stimulated uptake of radioactive (45)Ca(2+). Here, we first provide an overview of the mechanism of Ca(2+)-transport ATPases and show how this can be taken advantage of in protocols for measuring Ca(2+) pump activity.

The Ca2+ pumps of the endoplasmic reticulum and Golgi apparatus.

The various splice variants of the three SERCA- and the two SPCA-pump genes in higher vertebrates encode P-type ATPases of the P(2A) group found respectively in the membranes of the endoplasmic reticulum and the secretory pathway. Of these, SERCA2b and SPCA1a represent the housekeeping isoforms. The SERCA2b form is characterized by a luminal carboxy terminus imposing a higher affinity for cytosolic Ca(2+) compared to the other SERCAs. This is mediated by intramembrane and luminal interactions of this extension with the pump. Other known affinity modulators like phospholamban and sarcolipin decrease the affinity for Ca(2+). The number of proteins reported to interact with SERCA is rapidly growing. Here, we limit the discussion to those for which the interaction site with the ATPase is specified: HAX-1, calumenin, histidine-rich Ca(2+)-binding protein, and indirectly calreticulin, calnexin, and ERp57. The role of the phylogenetically older and structurally simpler SPCAs as transporters of Ca(2+), but also of Mn(2+), is also addressed.

Factors controlling the activity of the SERCA2a pump in the normal and failing heart.

Heart failure is the leading cause of death in western countries and is often associated with impaired Ca(2+) handling in the cardiomyocyte. In fact, cardiomyocyte relaxation and contraction are tightly controlled by the activity of the cardiac sarco(endo)plasmic reticulum (ER/SR) Ca(2+) pump SERCA2a, pumping Ca(2+) from the cytosol into the lumen of the ER/SR. This review addresses three important facets that control the SERCA2 activity in the heart. First, we focus on the alternative splicing of the SERCA2 messenger, which is strictly regulated in the developing heart. This splicing controls the formation of three SERCA2 splice variants with different enzymatic properties. Second, we will discuss the role and regulation of SERCA2a activity in the normal and failing heart. The two well-studied Ca(2+) affinity modulators phospholamban and sarcolipin control the activity of SERCA2a within a narrow window. An aberrantly high or low Ca(2+) affinity is often observed in and may even trigger cardiac failure. Correcting SERCA2a activity might therefore constitute a therapeutic approach to improve the contractility of the failing heart. Finally, we address the controversies and unanswered questions of other putative regulators of the cardiac Ca(2+) pump, such as sarcalumenin, HRC, S100A1, Bcl-2, HAX-1, calreticulin, calnexin, ERp57, IRS-1, and -2.