Structural Topology of SERCA-PLB Complex in Oriented Lipid Bicelles Detected by Multifrequency EPR

Abstract

We have used electron paramagnetic resonance (EPR) at X and Q bands to probe the structure of the integral membrane proteins phospholamban (PLB) and the sarcoplasmic reticulum calcium ATPase (SERCA). We found previously that PLB remains bound to SERCA after phosphorylation at S16, suggesting that a structural transition within the SERCA-PLB complex is responsible for relief of calcium transport inhibition (James & McCaffrey et al Biophys J 2012). Our current goal is to elucidate this structural mechanism through EPR on oriented bicelles, having previously optimized bicelle conditions to maximize protein alignment (McCaffrey et al JMR 2015), in order to support rational design of therapies to improve calcium transport in muscle cells. We used the rigid spin labels TOAC and BSL in conjunction with the monomeric mutant AFA-PLB and SERCA purified from rabbit skeletal muscle. These proteins were reconstituted into lipid bicelles and magnetically aligned at both X and Q bands, allowing for a robust global fitting approach. We measured significant changes in PLB transmembrane helix tilt upon phosphorylation, in the absence and presence of SERCA. Experiments were performed in the Biophysical Technology Center at the University of Minnesota. This work was funded by grants from NIH to DDT (R01 GM027906 and T32 AR007612).