Title: Phosphorylation Alters the Transmembrane Binding Interface Between Phospholamban and SERCA
Abstract: Cardiac muscle contraction is regulated through the release and reuptake of Ca2+ from the sarcoplasmic reticulum (SR), with the SR Ca2+-ATPase (SERCA) inducing relaxation by restoring Ca2+ to the SR lumen from the cytosol. SERCA is regulated by the single-pass membrane protein phospholamban (PLB), which acts as inhibitor unless phosphorylated at Ser16. The mechanism of SERCA-PLB regulation remains poorly understood, though our recent work demonstrated that PLB remains bound to SERCA following Ser16 phosphorylation, indicating that a structural rearrangement mediates inhibition relief. To further elucidate this mechanism, we have used site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy to study the interaction between the transmembrane (TM) domains of SERCA and PLB, where regulation is mediated. Spin labels were attached along PLB's TM helix for accessibility EPR measurements as well as double electron-electron resonance (DEER) distance measurements to spin-labeled SERCA. Our results suggest that Ser16 phosphorylation induces a change in the topology of PLB's TM helix that results in disruption of SERCA inhibition without dissociating the two proteins. Acknowledgments: spectroscopy was performed in the Biophysical Spectroscopy Center at the University of Minnesota, and funded by NIH grants to DDT (R01 GM27906, P30 AR507220).