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. 2025 Sep 5;64(18):4000–4010. doi: 10.1021/acs.biochem.5c00305

Structural Elements of Dwarf Open Reading Frame Required for Activation of the Sarco-Endoplasmic Reticulum Calcium Pump

M’Lynn E Fisher 1, Justin R Gregory 1, Stan T J Aanhane 1, M Joanne Lemieux 1, Howard S Young 1,*
PMCID: PMC12445340  PMID: 40910871

Abstract

The sarco-endoplasmic reticulum calcium pump (SERCA) is a P-type ATPase that plays a critical role in intracellular calcium signaling. SERCA maintains the calcium gradient between the cytosol and the sarco-endoplasmic reticulum, which is essential for a variety of physiological events including the muscle contraction-relaxation cycle. In cardiac muscle, SERCA is regulated by transmembrane peptides phospholamban (PLN) and dwarf open reading frame (DWORF). These peptides encode the opposing functions of SERCA inhibition by PLN and SERCA activation by DWORF, though the underlying mechanisms remain unclear. Herein, we investigated structural elements of DWORF expected to play a role in SERCA activation. We first measured SERCA activity in the absence and presence of DWORF variants targeting Leu12 and Pro15. These residues were selected based on sequence alignment with PLN. Leu12 and Pro15 of DWORF align with the essential residues Leu31 and Asn34 of PLN, which are required for SERCA inhibition. We found that both residues are required for SERCA activation by DWORF and that substitution of Pro15 (to Ala, Asn, or Leu) resulted in potent inhibition of SERCA. We next investigated the roles of Gly21, Ile23, and Gly25 in SERCA activation and DWORF oligomerization. These residues are part of a common helix interaction motif, GxxxG (Gly21-Trp-Ile-Val-Gly25) found in DWORF, which is unique among the regulins. The data suggest that the GxxxG motif does not play a role in DWORF oligomerization. Instead, this motif appears to interact with SERCA and provides a smooth interface that promotes activation and avoids inhibitory interactions with SERCA.

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Introduction

Intracellular signaling is an important mechanism by which cells respond to a variety of physiological cues. Calcium is one of the second messengers used to mediate intracellular signaling, which triggers life-enabling events such as insulin secretion, synaptic transmission, and muscle contraction. To enable such signaling events, a 2000-fold gradient is maintained between the low resting-state cytosolic calcium concentration and the high calcium concentration present in the sarco-endoplasmic reticulum (SR/ER). This gradient can be mobilized for signaling via inositol-1,4,5-triphosphate receptor (IP3R) or ryanodine receptor (RyR) calcium channels. These signaling mechanisms are essential for normal cell physiology, and disordered calcium handling underlies a diverse array of diseases including cardiomyopathies, skeletal muscle disorders, and neurological diseases. In cardiac muscle, SERCA maintains SR calcium stores, which is the major source of calcium for cardiac contractility. While SERCA calcium transport is essential for all cells, it is particularly important in cardiomyocytes where defects in SERCA activity or regulation are associated with cardiomyopathies and heart failure. − This connection with heart failure has focused attention on SERCA as a possible target for therapy. An initially promising approach in animal models, increasing SERCA expression via gene delivery, has proven challenging in human clinical trials. Nonetheless, the rationale for treating heart failure by improving calcium transport function remains compelling and novel approaches are required.

In the heart, SERCA activity is regulated to allow dynamic calcium homeostasis and cardiac contractility. A small transmembrane protein, phospholamban (PLN), is the primary regulatory subunit of SERCA in cardiac muscle. PLN inhibits SERCA by decreasing its apparent calcium affinity, reducing both the rate of calcium reuptake and the amount of calcium transported into the SR. PLN inhibition of SERCA thereby controls the rate of relaxation and the SR calcium load available for subsequent contractions, modulating both the lusitropic and inotropic properties of the heart. SERCA inhibition by PLN is relieved by phosphorylation via protein kinase A (PKA) and the β-adrenergic pathway. Under resting conditions, PLN is an important brake that prevents SR calcium overload and the arrhythmogenic consequences. In turn, regulation of PLN by phosphorylation creates a dynamic SR calcium load that can respond to the sympathetic need for increased cardiac output, the so-called “fight-or-flight” response. Thus, there is an extensive pool of SERCA pumps in the SR membrane and their combined calcium transport capacity can be finely tuned, either upregulated or downregulated, depending on the requirement for cardiac contractility.

For many decades, PLN was the only known regulatory subunit of SERCA in ventricular muscle. This changed with the discovery of another small transmembrane subunit of SERCA, dwarf open reading frame (DWORF). DWORF has weak sequence similarity to PLN (Figure ), and it was found to indirectly activate SERCA by displacing PLN and relieving inhibition. We subsequently discovered that DWORF directly activates SERCA by increasing its turnover rate, even in the absence of PLN. As an endogenous SERCA activator, DWORF represents an important new therapeutic target for the treatment of heart failure patients. In this study, we investigated the activation properties of DWORF using targeted mutagenesis, coreconstitution of wild-type and mutant forms of DWORF into proteoliposomes with SERCA, followed by measurement of calcium-dependent ATPase activity of SERCA. Based on sequence comparison to PLN, we targeted Leu12, Pro15, Gly21, Ile23, and Gly25 of DWORF. Leu12 and Pro15 of DWORF align with the two most important residues for PLN function, Leu31 and Asn34, respectively. , Gly21, Ile23, and Gly25 are part of a transmembrane helix–helix interaction motif, GxxxG (Gly21-Trp-Ile-Val-Gly25), that may play a role in the function or oligomerization of DWORF. We found that Pro15, Gly21, and Gly25 are essential for the structure and function of DWORF, where multiple variants studied resulted in loss of function and gain of SERCA inhibition. We suggest that Pro15 promotes a helix-linker-helix structure of DWORF that avoids inhibitory interactions with SERCA that are found in PLN. In addition, we suggest that Gly21 and Gly25 interact with SERCA and provide a smooth interface that promotes activation and avoids inhibitory interactions with SERCA. These structural elements of DWORF are optimized for SERCA activation and to avoid SERCA inhibition.

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Sequence alignment and topology diagram of DWORF and SERCA activation by DWORF. (A) Sequence alignments of phospholamban, sarcolipin, and DWORF. The predicted transmembrane domains (blue line) and residues targeted by mutagenesis (green) are indicated. (B) Topology diagrams of phospholamban, sarcolipin, and DWORF. The cytoplasmic (white), transmembrane (gray), and luminal (black) domains are indicated. The residues targeted by mutagenesis (green) are indicated. (C) Calcium-dependent ATPase activity data for SERCA in the absence and presence of wild-type DWORF. ATPase activity as a function of calcium concentration is shown for SERCA alone (solid gray line) and SERCA in the presence of wild-type DWORF (solid gray line). Each data point is the mean ± SEM (n ≥ 3). The V max, K Ca, and Hill coefficients (n H) are given in Table .

Materials and Methods

Materials

All reagents were of the highest purity available: octaethylene glycol monododecyl ether (C12E8; Barnet Products, Englewood Cliff, NJ); egg yolk phosphatidylcholine (EYPC), phosphatidylethanolamine (EYPE) and phosphatidic acid (EYPA) (Avanti Polar Lipids, Alabaster, AL); all reagents used in the coupled enzyme assay including NADH, ATP, PEP, lactate dehydrogenase, and pyruvate kinase (Sigma-Aldrich, Oakville, ON Canada).

Coreconstitution of DWORF and SERCA

Recombinant human DWORF (UniProt DWORF_HUMAN: P0DN84) was expressed as a maltose-binding protein (MBP) fusion with a TEV cleavage site for removal of MBP. DWORF was purified by a combination of organic extraction (chloroform-isopropanol-water) and reverse-phase HPLC. Purified DWORF was stored as lyophilized thin films (63 μg aliquots). SERCA1a was purified from rabbit skeletal muscle SR. Note that human and rabbit DWORF are 83% identical and human and rabbit SERCA1a are 84% identical. Importantly, the inhibitory groove of SERCA where DWORF binds , and the structural elements of DWORF investigated in this study are completely conserved.

For coreconstitution, lyophilized DWORF (63 μg) was suspended in a 100 μL mixture of trifluoroethanol–water (5:1) and mixed with lipids (360 μg EYPC & 40 μg EYPA) from stock chloroform solutions. The peptide-lipid mixture was dried to a thin film under nitrogen gas and desiccated under vacuum overnight. The peptide-lipid thin film was rehydrated in buffer (20 mM imidazole pH 7.0; 100 mM NaCl; 0.02% NaN3) at 50 °C for 15 min, cooled to room temperature, and detergent-solubilized by the addition of C12E8 (0.2% final concentration) with vigorous vortexing. Detergent-solubilized SERCA1a (UniProt AT2A1_RABIT: P04191) was added (300 μg in a total volume of 300 μL) and the reconstitution was stirred gently at room temperature. Detergent was slowly removed by the addition of SM-2 Bio-Beads (Bio-Rad, Hercules, CA) over a 4-h time course (final weight ratio of 25 Bio-Beads to 1 detergent). Following detergent removal, the reconstitution was centrifuged over a sucrose step-gradient (20% and 50% layers) for 1 h at 100,000g. The reconstituted proteoliposomes at the gradient interface were removed, flash-frozen in liquid-nitrogen, and stored at −80 °C. The final molar ratio was 120 lipids to 2 DWORF to 1 SERCA. To ensure the proper ratio of ∼2 DWORF per SERCA in the reconstituted proteoliposomes, quantitative SDS-PAGE was used with known quantities of SERCA and DWORF proteins as standards. ,,

ATPase Activity Assays of SERCA Reconstitutions

ATPase activity of the coreconstituted proteoliposomes was measured by a coupled-enzyme assay over a range of calcium concentrations from 0.1 μM to 10 μM. , The assay has been adapted to a 96-well format utilizing Synergy 4 (BioTek Instruments) or SpectraMax M3 (Molecular Devices) microplate readers. Data points were collected at 340 nm wavelength, with a well volume of 155 μL containing 10–20 nM SERCA at 30 °C (data points collected every 28–39 s for 1 h). The reactions were initiated by the addition of proteoliposomes to the assay solution. The V max (maximal activity) and K Ca (apparent calcium affinity) were determined based on nonlinear least-squares fitting of the activity data to the Hill equation (Sigma Plot software, SPSS Inc., Chicago, IL). Errors were calculated as the standard error of the mean (SEM) for a minimum of three independent reconstitutions. Statistical comparisons between groups were performed with the one-way ANOVA (between data sets) followed by the Holm-Sidak test for pairwise comparison. Differences were considered statistically significant at p < 0.05.

Denaturing Gel-Electrophoresis of DWORF

Tris-Tricine gels were run using a Bio-Rad Mini-PROTEAN system with gel-plate dimensions of 1 × 72 × 100 mm. Polyacrylamide gel layers were poured as follows: ∼57 mm height for a 16% separation gel, ∼10 mm height for a 10% spacer gel, and ∼15 mm height for a 4% stacking gel (gel buffer contained 1 M Tris, 0.1% w/v SDS, pH 8.45). Samples containing 0.1 to 0.5 μg of DWORF peptide were added to an equal volume of sample buffer (0.1 M Tris, 2% w/v SDS, 32% v/v glycerol, 0.1 M DTT, 0.15 mM bromophenol blue, pH 6.8) without heating. Gels were run the same day at 140 V until the dye front was 5 mm from the bottom of the gel (∼1 h; cathode buffer contained 0.1 M Tris, 0.1 M Tricine, 0.1% w/v SDS, pH 8.25; anode buffer contained 0.1 M Tris, pH 8.9). Proteins were visualized by silver staining as described with a 45 min incubation in fixing solution.

Results

SERCA Activity in the Presence of DWORF

Previous studies demonstrated that DWORF is a direct activator of SERCA. DWORF was found to increase the turnover rate and maximal activity (V max) of SERCA with no effect on the apparent calcium affinity (K Ca). Using the same experimental approach, wild-type DWORF was coreconstituted into membrane vesicles with SERCA and the calcium-dependent ATPase activity was measured (Figure ). As observed previously, DWORF increased the V max of SERCA ∼1.7-fold with no effect on the K Ca of SERCA (Table ). Thus, SERCA in the absence of DWORF served as a negative control and SERCA in the presence of wild-type human DWORF served as a positive control for all further experiments.

1. Kinetic Parameters for SERCA in the Absence and Presence of Wild-Type DWORF and DWORF Mutants.

  kinetic parameters
 number of independent reconstitutions
sample V max (μmol/min/mg) p value K Ca (μM calcium) p value n H p value n
SERCA 4.1 ± 0.1   ** 0.42 ± 0.03   ns 1.8 ± 0.2   ns 25
wtDWORF 6.9 ± 0.2 **   0.48 ± 0.03 ns   1.8 ± 0.2 ns   39
Leu12-Ala 5.2 ± 0.1 ** ** 0.42 ± 0.04 ns ns 1.4 ± 0.2 * ns 10
Pro15-Ala 2.1 ± 0.1 ** ** 0.73 ± 0.06 ** ** 1.2 ± 0.1 ** ** 10
Pro15-Asn 2.3 ± 0.1 ** ** 0.63 ± 0.07 ** * 2.4 ± 0.5 ns * 10
Pro15-Leu 3.5 ± 0.2 * ** 0.90 ± 0.17 ** ** 1.2 ± 0.2 ns ns 5
Gly21-Ala 6.8 ± 0.1 ** ns 1.38 ± 0.07 ** ** 1.1 ± 0.1 ns ns 15
Gly21-Phe 3.9 ± 0.1 ns ** 0.47 ± 0.03 ns ns 1.6 ± 0.1 ns ns 10
Gly21-Ile 3.7 ± 0.1 ns * 0.73 ± 0.04 ** ** 1.2 ± 0.1 ns ns 7
Ile23-Ala 5.7 ± 0.1 ** * 0.57 ± 0.04 ** * 1.6 ± 0.1 ns ns 9
Gly25-Ala 3.5 ± 0.1 * ** 0.87 ± 0.05 ** ** 1.2 ± 0.1 ns ns 17
Gly25-Phe 5.5 ± 0.1 ** ** 0.46 ± 0.02 ns ns 1.6 ± 0.1 ns ns 13
Gly25-Leu 3.5 ± 0.1 * ** 0.43 ± 0.02 ns ns 1.4 ± 0.1 ns ns 4
Gly21-Phe, Gly25-Phe 3.7 ± 0.1 ns ** 0.77 ± 0.07 ** ** 1.4 ± 0.2 ns ns 14
Gly25-Asp 4.5 ± 0.1 ns ** 0.66 ± 0.04 ns ns 1.0 ± 0.1 ns ns 3
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Statistical significance compared to SERCA reconstituted alone (left column) and SERCA reconstituted in the presence of wild-type DWORF (right column; ns, not significant; * p < 0.05; ** p < 0.01).

Targeted Mutagenesis of DWORF Pro15

We previously suggested that Pro15 is an important residue for DWORF function based on sequence alignments with PLN and SLN. , Asn34 of PLN and Asn11 of SLN are essential for inhibition of SERCA, and these residues align with Pro15 of DWORF. Considering the opposing functions of SERCA inhibition by PLN and activation by DWORF, Pro15 appeared to be an important structural element of DWORF. We tested the role of this residue by first mutating Pro15 to asparagine and we hypothesized that the activation behavior of wild-type DWORF should convert to inhibitory behavior for the Pro15-Asn substitution. Pro15-Asn DWORF was coreconstituted into proteoliposomes with SERCA and the calcium-dependent ATPase activity was measured (Figure ). We observed superinhibition of SERCA in the presence of Pro15-Asn DWORF. Compared to SERCA in the absence and presence of wild-type DWORF, the maximal activity of SERCA was strongly suppressed in the presence of Pro15-Asn DWORF (V max values of 4.1, 6.9, and 2.4 μmol/min/mg, respectively). The apparent calcium affinity of SERCA was also decreased in the presence of the Pro15-Asn variant (K Ca values of 0.63 μM calcium), compared to SERCA in the absence and presence of wild-type DWORF (K Ca values of 0.42 and 0.48 μM calcium, respectively). As hypothesized, Pro15-Asn DWORF inhibited SERCA, though the observed superinhibition was unexpected (Table ). Interestingly, the Pro15-Asn variant of DWORF increased the cooperativity for calcium binding, which is a characteristic feature of PLN. Pro15-Asn DWORF altered the Hill coefficient and increased the cooperativity for calcium binding to SERCA compared to wild-type DWORF (n H of 2.4 ± 0.5 compared to 1.8 ± 0.2).

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Calcium-dependent ATPase activity data for SERCA in the presence of DWORF mutants Pro15-Asn, Pro15-Ala, and Pro15-Leu (dashed lines). SERCA in the absence and presence of wild-type DWORF are shown for comparison (light gray; data from Figure C). Each data point is the mean ± SEM (n ≥ 3). The Vmax, KCa, and Hill coefficients (n H) are given in Table .

Two additional substitutions were also tested at this position, Pro15-Ala and Pro15-Leu (Figure ). This latter variant has been identified through genetic testing with unknown clinical significance (dbSNP rs1277330938). Based on our results, both variants can be considered superinhibitors of SERCA, though the impact on SERCA kinetic parameters is different. Like the Pro15-Asn DWORF variant, the Pro15-Ala and Pro15-Leu variants impacted both the K Ca and V max of SERCA (Table ). The maximal activity of SERCA was suppressed in the presence of the Pro15-Ala and Pro15-Leu variants of DWORF (V max values of 2.4 and 3.5 μmol/min/mg, respectively), as compared to SERCA in the absence and presence of wild-type DWORF (V max values of 4.1 and 6.9 μmol/min/mg, respectively). The apparent calcium affinity of SERCA was also decreased in the presence of the Pro15-Ala and Pro15-Leu variants (K Ca values of 0.74 and 0.91 μM calcium, respectively), compared to SERCA in the absence and presence of wild-type DWORF (K Ca values of 0.42 and 0.48 μM calcium, respectively). Compared to wild-type and Pro15-Asn DWORF, the Pro15-Ala and Pro15-Leu variants decreased the cooperativity for calcium binding to SERCA (n H 1.2 ± 0.1 and 1.2 ± 0.2, compared to 1.8 ± 0.2 and compared to 2.4 ± 0.5, respectively). Thus, all three mutants, Pro15-Asn, Pro15-Ala, and Pro15-Leu, were potent inhibitors of SERCA that impacted both the V max and K Ca of SERCA.

Targeted Mutagenesis of DWORF Leu12

With the superinhibitory characteristics of the Pro15 mutants described above, this suggested that the sequence alignment between PLN, SLN, and DWORF was correct. To further test this, we focused on Leu12 of DWORF which is predicted to align with Leu31 of PLN and Leu8 of SLN. Together, Leu31 and Asn34 of PLN and Leu8 and Asn11 of SLN are essential residues for inhibition of SERCA, as alanine substitution of any of these residues results in complete loss of function. ,, Thus, the Leu-x-x-Asn sequence (Figure ) seems to be an important structural element for PLN and SLN inhibition of SERCA. Since DWORF is an activator of SERCA, not an inhibitor, we wondered if Leu12 would also be important for SERCA activation by DWORF. SERCA was coreconstituted into proteoliposomes in the absence and presence of Leu12-Ala DWORF, which resulted in partial loss of activation (Figure and Table ). The V max value for SERCA in the presence of Leu12-Ala DWORF was 5.0 μmol/min/mg. This value was close to that determined for SERCA alone (4.1 μmol/min/mg) and distinct from that determined for SERCA in the presence of wild-type DWORF (6.9 μmol/min/mg). Thus, Leu12 of DWORF appears to be important for SERCA activation, mirroring the importance of Leu31 and Leu8 in SERCA inhibition by PLN and SLN. In addition, the inhibitory dyad of Leu31 and Asn34 in PLN and Leu8 and Asn11 in SLN appears to be repurposed as a Leu12 and Pro15 dyad in DWORF for SERCA activation.

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Calcium-dependent ATPase activity data for SERCA in the presence of DWORF mutants Leu12-Ala, Gly21-Ala, and Gly 25-Ala (dashed lines). SERCA in the absence and presence of wild-type DWORF are shown for comparison (light gray; data from Figure C). Each data point is the mean ± SEM (n ≥ 3). The V max, K Ca, and Hill coefficients (n H) are given in Table .

Targeted Mutagenesis of DWORF Gly21, Ile23, and Gly25

A seemingly unique structural element of DWORF is the GxxxG motif consisting of residues Gly21-Trp22-Ile23-Val24-Gly25 (Figure ). The GxxxG motif was originally identified as a dimerization motif in glycophorin A, though we now know it is a common sequence of residues that favors helix–helix interactions in membrane proteins. The GxxxG sequence in DWORF aligns with Ile40-Cys41-Leu42-Leu43-Leu44 in PLN. This region of PLN is important for oligomerization, where Ile40 and Leu44 are part of a leucine-isoleucine zipper that stabilizes the PLN pentamer. , Interestingly, Ile40 and Leu44 of PLN align with Gly21 and Gly25 of DWORF, suggesting that PLN and DWORF may have distinct oligomerization motifs localized to the same region of the peptides. Therefore, we hypothesized that the Gly21-Trp22-Ile23-Val24-Gly25 sequence is important for DWORF function and oligomerization.

We first examined the functional importance of Gly21, Gly25, as well as Ile23 in the center of this motif. Ile23 of DWORF aligns with Leu42 of PLN (Figure ), which is another residue involved in PLN inhibition of SERCA. , A Leu42-Ala substitution in PLN results in partial loss of function. ,, SERCA was coreconstituted into proteoliposomes in the absence and presence of Gly21-Ala, Ile23-Ala, and Gly25-Ala mutants of DWORF (Figures and ). Another variant, Gly25-Asp, was identified through genetic testing (dbSNP rs1200203728; Figure ). The calcium-dependent ATPase activity assays indicated that the maximal activity (V max) of SERCA in the presence of the Gly21-Ala mutant was similar to wild-type DWORF, though Gly21-Ala gained inhibitory function by decreasing the apparent calcium affinity of SERCA (Table ). The K Ca values for SERCA in the presence of wild-type and Gly21-Ala DWORF were 0.48 and 1.38 μM calcium, respectively. The Ile23-Ala mutant resulted in partial loss of function, with V max and K Ca values that were similar to SERCA in the presence of wild-type DWORF (Table ; K Ca values of 0.57 and 0.48 μM calcium, respectively; V max values of 5.7 and 6.9 μmol/mg/min, respectively). The final two mutants, Gly25-Ala and Gly25-Asp, emphasized the importance of this particular residue in tuning the balance between SERCA activation and SERCA inhibition. Both mutants were inhibitory, with SERCA K Ca values of 0.87 and 0.66 μM calcium, respectively (compared to 0.48 μM calcium for SERCA in the presence of wild-type DWORF). Similarly, the V max values indicated lack of activation of SERCA by Gly25-Asp DWORF (4.5 μmol/min/mg) and inhibition of SERCA by Gly25-Ala DWORF (3.5 μmol/min/mg). Thus, Gly21 and Gly25 were clearly important for DWORF activation behavior and to avoid inhibitory interactions with SERCA.

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Calcium-dependent ATPase activity data for SERCA in the presence of DWORF mutants Gly25-Asp and Ile23-Ala (dashed lines). SERCA in the absence and presence of wild-type DWORF are shown for comparison (light gray; data from Figure C). Each data point is the mean ± SEM (n ≥ 3). The V max, K Ca, and Hill coefficients (n H) are given in Table .

The Gly21-Ala and Gly25-Ala mutants demonstrated the sensitivity of DWORF and SERCA to amino acid substitution at these positions. However, the GxxxG transmembrane helix interaction motif is also known as the (small)­xxx­(small) motif because of the common occurrence of other small residues such as alanine and serine. To further understand the role in glycines in SERCA activation by DWORF, we generated phenylalanine substitutions of each residue (Gly21-Phe and Gly25-Phe), as well as the double mutant (Gly21-Phe, Gly25-Phe). The Gly21-Phe mutant resulted in complete loss of function, with V max and K Ca values that were similar to SERCA alone (Figure and Table ). Interestingly, the Gly25-Phe mutant retained the ability to activate SERCA and was most similar to SERCA in the presence of wild-type DWORF (V max values of 5.5 and 6.9 μmol/min/mg; K Ca values of 0.46 and 0.48 μM calcium, respectively). Finally, the double mutant Gly21-Phe, Gly25-Phe resulted in complete loss of function compared to wild-type DWORF (V max values of 3.7 and 6.9 μmol/min/mg, respectively) and gain of inhibitory function (K Ca values of 0.77 and 0.48 μM calcium, respectively). Considering these data, the overall trend for the alanine substitutions of Gly21 and Gly25 was gain of SERCA inhibition, while the trend for the phenylalanine substitutions was loss of SERCA activation.

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Calcium-dependent ATPase activity data for SERCA in the presence of DWORF mutants Gly21-Phe and Gly25-Phe and the double mutant Gly21-Phe, Gly25-Phe (dashed lines). SERCA in the absence and presence of wild-type DWORF are shown for comparison (light gray; data from Figure C). Each data point is the mean ± SEM (n ≥ 3). The V max, K Ca, and Hill coefficients (n H) are given in Table .

Oligomerization of DWORF Mutants

Our initial hypothesis was that the GxxxG motif of DWORF contributes to oligomer formation. SDS-PAGE has been widely used as an indicator of PLN oligomerization, with PLN monomers and pentamers (and sometimes dimers, trimers and tetramers) clearly visible based on electrophoretic mobility. In particular, SDS-PAGE has been used as a facile readout for changes in PLN oligomerization caused by mutations (e.g., alanine-scanning mutagenesis of PLN , ). To examine the oligomeric state of DWORF, we performed SDS-PAGE and examined the electrophoretic behavior of the peptide (Figure ). Wild-type DWORF and the mutants described above were subjected to SDS-PAGE on a 16% gel under denaturing conditions. All peptides formed two main species observable by SDS-PAGE and silver staining, which were assumed to be monomers and dimers of DWORF. However, weaker additional bands were observed that suggested lower- and higher-order oligomeric species. In order to characterize a monomeric form of DWORF, we synthesized a peptide with a scrambled amino acid sequence with the assumption that it would not form oligomers. The scrambled peptide migrated close to the correct molecular weight of ∼3.7 kDa by SDS-PAGE (scramb in Figure A). This migration behavior for the scrambled peptide indicated that the two prominent bands observed for DWORF likely corresponded to a dimer and tetramer. In support of this conclusion, one of the DWORF mutants studied, Gly25-Ala, migrated as a ladder of bands consistent with a monomer, dimer, trimer, tetramer, pentamer, and hexamer (G25A in Figure B).

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Sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) of DWORF variants. (A) Gels are shown for scrambled DWORF peptide (scram), wild-type DWORF (WT), DWORF mutants Leu12-Ala (L12A), Pro15-Ala (P15A), Pro15-Leu (P15L), Pro15-Asn (P15N), Gly21-Ala (G21A), Gly21-Phe (G21F), Ile23-Ala (I23A), Gly25-Ala (G25A), Gly25-Phe (G25F), and Gly25-Asp (G25D), and the double mutant Gly21-Phe, Gly25-Phe (G21F/G25F). The bars on the right indicate the positions of the dimer and tetramer. A minimum of six SDS-PAGE experiments were performed for each variant and a representative gel was chosen. (B) Integration of the gel for the Gly25-Ala variant showing a series of oligomeric species including a monomer, dimer, trimer, tetramer, pentamer, and a faint hexamer.

With this interpretation, all DWORF variants in the present study were capable of forming dimers. However, some of the mutant peptides exhibited slight variations in migration behavior compared to wild-type DWORF, with the most notable being Gly25-Asp (G25D in Figure A). Our interpretation of this variant is that it still forms dimers and tetramers, though the migration behavior is faster (smaller apparent molecular weight) due to the added negative charge. We have previously observed anomalous migration behavior for peptides by SDS-PAGE, as well as changes in migration behavior with amino acid substitution. Nonetheless, the Gly21 and Gly25 substitutions were still capable of forming dimers, as well as tetramers in most cases. Even the double phenylalanine substitution (Gly21-Phe, Gly25-Phe; G21F/G25F in Figure A) was capable of forming stable dimers by SDS-PAGE. Thus, the GxxxG motif of DWORF did not appear to be a major contributor to oligomerization. Surprisingly, the Ile23-Ala variant appeared to yield the most monomers by SDS-PAGE (I23A in Figure A), as well as general destabilization of the dimer and tetramer. This suggested that a different oligomerization motif may be involved in DWORF oligomerization.

Mimicking PLN

The substitution of Ile23 had the largest impact on DWORF dimerization, suggesting that DWORF may possess a different oligomerization motif. To begin to test this, we substituted the glycine residues of DWORF with the corresponding residues in PLN. Gly21 and Gly25 in DWORF align with Ile40 and Leu44 in PLN (Figure ). Ile40 and Leu44 have been implicated in the Leu-Ile zipper that stabilizes the pentameric from of PLN. We generated Gly21-Ile and Gly25-Leu variants of DWORF and first tested their ability to activate SERCA (Figure ). The Gly21-Ile variant resulted in loss of SERCA activation and gain of SERCA inhibition, and the Gly25-Leu variant resulted in complete loss of function (Table ). We next tested the oligomeric state of these variants as observed by SDS-PAGE (Figure , insets). Interestingly, both variants formed a stable dimer and tetramer, with no detectable monomer compared to wild-type DWORF (Figure A).

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Calcium-dependent ATPase activity data for SERCA in the presence of DWORF mutants Gly21-Ile and Gly25-Leu (dashed lines) to emulate the residues found in phospholamban. SERCA in the absence and presence of wild-type DWORF are shown for comparison (light gray; data from Figure C). Each data point is the mean ± SEM (n ≥ 3). The V max, K Ca, and Hill coefficients (n H) are given in Table . Insets: SDS-PAGE of the Gly21-Ile and Gly25-Leu DWORF variants revealing dimer and tetramer bands and the absence of a monomer band. Comparing the insets to wild-type DWORF (WT) in Figure A.

Discussion

Pro15 Is Essential for SERCA Activation

SERCA activity is altered by a wide variety of highly divergent transmembrane peptides including the regulins, sarcolambans, and even model peptides. , Most peptides are inhibitory and the most common feature is a reduction in the maximal activity of SERCA. Many peptides also alter the apparent calcium affinity of SERCA, with PLN and SLN being well-characterized examples. Therefore, as a transmembrane peptide regulator of SERCA, DWORF stands out as the only known activator of SERCA–i.e., DWORF increases the maximal activity of SERCA without altering the apparent calcium affinity. This unique property of DWORF has garnered interest as a gene therapy for the treatment of cardiomyopathies and muscle wasting diseases.

In the present study, our goal was to identify structural elements of DWORF that are required for SERCA activation. Based on a sequence alignment (Figure and ref ) and previous studies of DWORF structure and function, , we focused on Pro15 as an essential structural feature of DWORF. Pro15 of DWORF induces a helix-linker-helix structure of DWORF that plays a major role in SERCA activation. In PLN and SLN, this residue is an asparagine that is essential for SERCA inhibition (Asn34 and Asn11, respectively). Thus, the presence of Pro15 in DWORF removes an inhibitory interaction found in PLN and SLN and replaces it with a structural element conducive for SERCA activation. The first variant tested was a Pro15-Asn mutant of DWORF that was expected to become a PLN-like inhibitor of SERCA (Figure ). While this mutant of DWORF was found to inhibit SERCA, the inhibitory effect was on the maximal activity (V max) of SERCA with a small effect on the apparent calcium affinity (K Ca). Thus, Pro15-Asn DWORF gained inhibitory function, but it was distinct from SERCA inhibition by PLN (e.g., refs ,, ). To further explore this residue, we generated the reciprocal mutant of PLN, Asn34-Pro, which resulted in complete loss of PLN function (data not shown). Thus, Pro15 is critical for DWORF function, though it is not the only structural element that differentiates SERCA activation by DWORF from SERCA inhibition by PLN.

We next generated Pro15-Ala and Pro15-Leu mutants of DWORF based on a standard alanine substitution and a human genetic variant, respectively. These variants were potent inhibitors of SERCA, altering both the maximal activity (V max) and apparent calcium affinity (K Ca) of SERCA (Figure ). This was consistent with the role of Pro15 in promoting an appropriate structure of DWORF for SERCA activation, where the variants tested eliminated activation and promoted SERCA inhibition. It is instructive to consider these residues–proline, alanine, and leucine–in terms of their propensity to promote secondary structure in DWORF. Alanine and leucine have the highest propensity to form an α-helix, while proline has the lowest propensity. This suggests that Pro15-Ala and Pro15-Leu may be more helical forms of DWORF. To test this hypothesis, we used AlphaFold to generate structural models of wild-type, Pro15-Asn, Pro15-Ala, and Pro15-Leu DWORF (Figure ). All Pro15 variants of DWORF were predicted to be more helical than wild-type DWORF supporting the concept that the inhibitory variants are more helical. In the crystal structure of the SERCA-PLN inhibitory complex, this region of PLN is helical. Thus, a more helical form of DWORF emulates the structure of PLN and results in SERCA inhibition.

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AlphaFold prediction models of wild-type DWORF (cyan) and the mutants Pro15-Asn (magenta), Pro15-Ala (green), and Pro15-Leu (yellow). The positions of Pro15 and the N- and C-termini of wild-type DWORF are indicated. Note that the three mutants are more helical forms of DWORF from Ala5 to the C-terminus. AlphaFold predicted the N-terminus of DWORF to be disordered in the mutants.

GxxxG Motif of DWORF Interacts with SERCA

DWORF is thought to bind to the inhibitory groove of SERCA and displace PLN, where the inhibitory groove is formed by transmembrane segments M2, M6 and M9. However, in binding to the inhibitory groove of SERCA, DWORF must avoid interactions that contribute to SERCA inhibition. , In part, this is facilitated by Pro15 and the helix-linker-helix structure of DWORF. Compared to the PLN inhibitory interactions of Leu31 and Asn34 with SERCA, the corresponding residues of Leu12 and Pro15 in DWORF do not interact with SERCA. Nonetheless, the transmembrane helix of DWORF binds to the inhibitory groove , and a large number of nonspecific transmembrane peptides are known to inhibit SERCA. , This suggested that there must be additional structural elements that dictate how DWORF interacts with the inhibitory groove while avoiding SERCA inhibition. A candidate helix–helix interaction motif in DWORF that we wished to understand was the GxxxG motif, Gly21-Trp-Ile-Val-Gly25 (Figure ). The GxxxG motif, or (small)­xxx­(small) motif, is a common sequence of residues found to mediate helix–helix interactions in membrane proteins. Much of what we know about this motif comes from the classic example of the glycophorin A transmembrane dimer. A combination of biochemical and structural studies revealed the role of the glycine residues in dimer formation, where they provided a smooth interface that allows close packing of the transmembrane α-helices. ,, Interestingly, the transmembrane domain of glycophorin A forms dimers observable by SDS-PAGE and the dimers can be disrupted by mutagenesis.

With this in mind, we expected that the GxxxG motif may play a role in DWORF dimerization and SERCA activation. We also expected that dimer formation would be sensitive to mutation and observable by SDS-PAGE. We generated alanine and phenylalanine variants of Gly21 and Gly25 and tested their ability to form oligomeric species (Figure A). Unlike glycophorin A and PLN, the DWORF variants did not significantly alter the migration pattern by SDS-PAGE, with dimers and tetramers being the prevalent species observed. Since the DWORF variants in the GxxxG motif did not significantly impact the oligomeric state of DWORF, we considered their effects on SERCA activation. Overall, the variants had a greater impact on DWORF function and SERCA activation (Table ) than they did on the oligomeric state of DWORF (Figure ). The alanine and phenylalanine variants of Gly21 and Gly25 resulted in either loss of DWORF function or gain of SERCA inhibition (Figures and ), indicating that even the substitution of another small amino acid was not tolerated. Importantly, the gain of inhibition observed for the Gly21-Ala and Gly25-Ala variants suggested that the alanine substitutions create additional contacts with SERCA that resulted in inhibition. Comparing the effects of the alanine and phenylalanine substitutions on the V max of SERCA revealed opposite effects for Gly21 and Gly25 of DWORF. The Gly21-Ala and Gly25-Phe variants of DWORF retained the ability to activate SERCA (increased V max; Table ), while the Gly21-Phe and Gly25-Ala variants of DWORF lost the ability to activate SERCA (decreased V max). The double mutant, Gly21-Phe and Gly25-Phe, most closely resembled the Gly21-Phe variant. These different requirements for Gly21 and Gly25 of DWORF likely reflect distinct interactions with SERCA and a dominant role for Gly21. Overall, the data suggests that the GxxxG motif of DWORF interacts with SERCA. In combination with the helix-linker-helix structure of DWORF promoted by Pro15, the smooth interface provided by the GxxxG motif avoids inhibitory interactions with SERCA.

Oligomerization of DWORF

While the GxxxG motif did not appear to play a role in DWORF oligomerization, one of the variants studied exhibited more DWORF monomers by SDS-PAGE (Figure ). Compared to wild-type DWORF, the Ile23-Ala variant appeared to destabilize the dimer and tetramer and yielded more monomers. This unexpected result led us to consider that DWORF may contain a Leu-Ile zipper motif like PLN. While the focus of this study was not on the leucine and isoleucine residues of DWORF, we nonetheless generated Gly21-Ile and Gly25-Leu variants to further investigate the behavior of the Ile23-Ala variant. Based on the sequence alignment (Figure ), Gly21 and Gly25 of DWORF correspond to Ile40 and Leu44 of PLN. These latter residues are involved in PLN pentamer formation. As evaluated by SDS-PAGE, both variants were similar to wild-type DWORF except for the absence of a band corresponding to the monomer (Figure ). This suggested that the main oligomeric states of DWORF, dimers and tetramers, were stabilized by these substitutions. In the functional assays, the Gly21-Ile and Gly25-Leu variants had a larger impact on DWORF function, which was comparable to the other glycine variants studied. The Gly21-Ile variant resulted in SERCA inhibition and the Gly25-Leu variant resulted in loss of function (Table ). These results support the notion that the GxxxG motif of DWORF interacts with SERCA and that different structural elements are responsible for the oligomerization of DWORF.

Conclusions

In the present study, five residues of DWORF were targeted for mutagenesis and 13 variants were tested for their ability to activate SERCA and self-associate into DWORF oligomers. Structural elements of DWORF that are important for SERCA activation versus inhibition were inferred based on assessment of SERCA activity, denaturing gel-electrophoresis of DWORF, and structural predictions of DWORF. Our goal was to identify residues that mediate SERCA–DWORF or DWORF–DWORF interactions. In this context, the results implicate Pro15 and the GxxxG motif as structure elements of DWORF that promote SERCA activation. The GxxxG motif of DWORF did not appear to contribute to oligomerization. Instead, the SERCA-DWORF interface was found to be exquisitely sensitive to changes in structure. Changes in the helical structure of DWORF (e.g., Pro15-Ala) or small changes in side chain structure (e.g., Gly21-Ala) resulted in potent inhibition of SERCA. We conclude that the sequence and structure of DWORF are optimized for SERCA activation and to avoid SERCA inhibition.

Acknowledgments

This work was funded by the Heart and Stroke Foundation of Canada (to H.S.Y.) and the Natural Sciences and Engineering Research Council of Canada (549297-2019 to M.J.L.).

DWORF_HUMAN: P0DN84, AT2A1_RABIT: P04191

†.

Authors contributed equally.

The authors declare no competing financial interest.

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