A hallmark of phospholamban functional divergence is located in the N-terminal phosphorylation domain

Sarcoplasmic reticulum Ca²⁺ pump (SERCA) is a critical component of the Ca²⁺ transport machinery in myocytes. There is clear evidence for regulation of SERCA activity by PLB, whose activity is modulated by phosphorylation of its N-terminal domain (residues 1–25), but there is less clear evidence for the role of this domain in PLB's functional divergence. It is widely accepted that only sarcolipin (SLN), a protein that shares substantial homology with PLB, uncouples SERCA Ca²⁺ transport from ATP hydrolysis by inducing a structural change of its energy-transduction domain; yet, experimental evidence shows that the transmembrane domain of PLB (residues 26–52, PLB_26–52) partially uncouples SERCA in vitro. These apparently conflicting mechanisms suggest that PLB's uncoupling activity is encoded in its transmembrane domain, and that it is controlled by the N-terminal phosphorylation domain. To test this hypothesis, we performed molecular dynamics simulations (MDS) of the binary complex between PLB_26–52 and SERCA. Comparison between PLB_26–52 and wild-type PLB (PLB_WT) showed no significant changes in the stability and orientation of the transmembrane helix, indicating that PLB_26–52 forms a native-like complex with SERCA. MDS showed that PLB_26–52 produces key intermolecular contacts and structural changes required for inhibition, in agreement with studies showing that PLB_26–52 inhibits SERCA. However, deletion of the N-terminal phosphorylation domain facilitates an order-to-disorder shift in the energy-transduction domain associated with uncoupling of SERCA, albeit weaker than that induced by SLN. This mechanistic evidence reveals that the N-terminal phosphorylation domain of PLB is a primary contributor to the functional divergence among homologous SERCA regulators.