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

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.