Molecular Details of SERCA Regulation by Phospholamban Revealed by Paramagnetic Relaxation Enhancements and Solid-State NMR

Abstract

Sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) ia a 110 kDa membrane protein that regulates cardiomyocyte relaxation by transporting Ca2+ into the SR. SERCA is regulated by PLN, a 52-resdiue membrane protein consisting of a helical, inhibitory transmembrane domain and a regulatory cytoplasmic domain. The cytoplasmic domain is in equilibrium between a folded T state and an unfoled R state. Phosphorylation of PLN at S16 relieves the inhibition of SERCA and, based on studies in animal models, mimicking the effects of PLN phosphorylation is a promising path for the development of therapeutics against heart failure.Here we co-reconstituted SERCA and PLN in lipid bilayers under fully functional conditions and utilized solid state NMR to probe the molecular mechanisms of SERCA regulation. Asymmetric spin labeling and isotopic labeling of SERCA, PLN and lipids in combination with magic angle spinning experiments provided paramagnetic relaxation enhancements (PREs) that mapped the structure of the membrane protein complex. PREs were combined with oriented restraints from separated local field experiments, chemical shift perturbations and torsion angles from backbone chemical shifts to determine a structural model of the PLN/SERCA complex. The transmembrane domain of PLN remains anchored to the ATPase, while the cytoplasmic domain is membrane-associated in the T state and interacts transiently with the phosphorylation and nucleotide domains of SERCA in the R state. Phosphorylation increases the R state population and the affinity of the R state for SERCA. These results explain the non-inhibitory character of PLN mutants with increased R state population and provide crucial information for future design of therapeutics to increase SERCA function. In addition, the methodological advances described here further open the landscape to conducting structural studies of membrane protein complexes of molecular weights beyond 100 kDa.