Distributed Structural Elements of DWORF Coordinate Activation of the Sarcoplasmic Reticulum Calcium Pump SERCA

Abstract Many physiological processes are controlled by changes in cytosolic calcium, including muscle contraction, neuronal transmission, and cell proliferation. In muscle, the sarco-endoplasmic reticulum calcium ATPase (SERCA) restores resting cytosolic calcium by transporting calcium into the sarcoplasmic reticulum, thereby promoting relaxation. In cardiac muscle, SERCA is regulated by phospholamban (PLN) and dwarf open reading frame (DWORF), which inhibit and activate SERCA, respectively. To investigate the mechanism of DWORF-mediated activation, we focused on Trp22, an unusual tryptophan residue positioned near the center of the DWORF transmembrane domain. This position resembles the recently identified transmembrane binding site for small-molecule SERCA activators, suggesting that Trp22 may play a direct role in activation. Consistent with this hypothesis, calcium-dependent ATPase measurements in reconstituted membranes showed that a Trp22-Ala substitution completely abolished SERCA activation. We next tested whether residues outside the central transmembrane region contribute to activation by modulating SERCA-DWORF interactions. Phosphomimetic substitutions at the N-terminal residues Ser7, Thr8, and Ser10 eliminated SERCA activation, suggesting that the N-terminus modulates DWORF positioning. More strikingly, substitutions within the C-terminal motif Tyr31, Val32, Phe34, and Ser35 profoundly altered function. Tyr31-Ala, Val32-Ala, and Phe34-Ala converted DWORF from an activator to an inhibitor, whereas Ser35-Ala and Ser35-Pro caused loss of activation. Together, these results support a coordinated mechanism in which Trp22 acts as a critical determinant of SERCA activation, the N-terminus provides the potential for reversible regulation, and the C-terminus constrains the DWORF transmembrane helix in an activation-competent state.