The mechanism of dwarf open reading frame (DWORF) activation of the sarcoplasmic reticulum calcium pump SERCA

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitory transmembrane peptide phospholamban. A new transmembrane peptide regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. We have shown that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban (1). Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake, while phospholamban decreases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium, while phospholamban has a higher affinity for SERCA in the absence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium. Mutagenesis and structure-function analysis of DWORF reveals three important features required for SERCA activation: Pro15 promotes a helix-linker-helix structure of DWORF; the N-terminal amphipathic helix of DWORF perturbs the membrane bilayer; and the short transmembrane helix of DWORF contributes to hydrophobic mismatch and positions the amphipathic helix at the surface of the membrane bilayer. Our current understanding of the mechanism of SERCA activation by DWORF will be presented.