Characterizing the Phospholamban-SERCA Complex by Pulsed EPR Spectroscopy

Abstract

Muscle contraction and relaxation are controlled through the release and reuptake of Ca2+ stored in the sarcoplasmic reticulum (SR). Relaxation is mediated by the SR Ca2+ ATPase (SERCA), a pump that drives Ca2+ against its concentration gradient while hydrolyzing ATP. Cardiac SERCA is regulated by phospholamban (PLB), a small membrane protein that inhibits the pump except when phosphorylated at Ser16. PLB phosphorylation restores SERCA activity without dissociating the two proteins, instead inducing a structural change within the PLB-SERCA complex. Although a number of studies have investigated the interaction of these proteins, the relationship between phosphorylation, structure, and activity remains unresolved. We have used dipolar electron-electron resonance (DEER) spectroscopy, a technique capable of measuring distances from 2-7nm, to characterize large conformational changes within PLB upon phosphorylation and SERCA binding. Our results show that the transmembrane and cytoplasmic helices of PLB draw closer upon SERCA binding, with subsequent phosphorylation compacting the structure still further. However, relative distances between the cytoplasmic domains of PLB and SERCA remain largely constant before and after phosphorylation, suggesting that the observed structural change occurs in the transmembrane domain of PLB. Ultimately, our goal is to make exhaustive distance measurements within the SERCA-PLB complex in order to understand the structural basis of phosphorylation-mediated inhibition relief.