Phosphorylation-dependent Conformational Switch in Spin-labeled Phospholamban Bound to SERCA

Christine B. Karim, Zhiwen Zhang, Edmund C. Howard, Kurt D. Torgersen, David D. Thomas

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We have used chemical synthesis, functional reconstitution, and electron paramagnetic resonance (EPR) to probe the functional dynamics of phospholamban (PLB), which regulates the Ca-ATPase (SERCA) in cardiac sarcoplasmic reticulum. The transmembrane domain of PLB inhibits SERCA at low [Ca2+], but the cytoplasmic domain relieves this inhibition upon Ser16 phosphorylation. Monomeric PLB was synthesized with Ala11 replaced by the 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) spin label, which reports peptide backbone dynamics directly. PLB was reconstituted into membranes in the presence or absence of SERCA. TOAC-PLB showed normal inhibitory function, which was reversed by phosphorylation at Ser16 or by micromolar [Ca2+]. EPR showed that the PLB cytoplasmic domain exhibits two resolved conformations, a tense T state that is ordered and a relaxed R state that is dynamically disordered and extended. PLB phosphorylation shifts this equilibrium toward the R state and makes it more dynamic (hyperextended). Phosphorylation strongly perturbs the dynamics of SERCA-bound PLB without dissociating the complex, while micromolar [Ca2+] has no effect on PLB dynamics. A lipid anchor synthetically attached to the N terminus of PLB permits Ca-dependent SERCA inhibition but prevents the phosphorylation-induced disordering and reversal of inhibition. We conclude that the relief of SERCA inhibition by PLB phosphorylation is due to an order-to-disorder transition in the cytoplasmic domain of PLB, which allows this domain to extend above the membrane surface and induce a structural change in the cytoplasmic domain of SERCA. This mechanism is distinct from the one that relieves PLB-dependent SERCA inhibition upon the addition of micromolar [Ca2+].

Original languageEnglish (US)
Pages (from-to)1032-1040
Number of pages9
JournalJournal of Molecular Biology
Issue number4
StatePublished - May 12 2006

Bibliographical note

Funding Information:
This work was supported by grants to D.D.T. (NIH GM27906) and C.B.K. (AHA 9930083N). Supercomputer time was provided by the Minnesota Supercomputing Institute. We thank Razvan Cornea, Gianluigi Veglia and Nathaniel Traaseth for insightful discussions; Deborah Winters for assistance in molecular modeling and graphics; Yuri Nesmelov for assistance with EPR spectroscopy; Florentin Nitu for technical assistance; Nathan Lockwood for providing the lipid anchor; Thomas Krick for assistance with mass spectrometry; Jinny Johnson and Lawrence Dangott (Protein Chemistry Laboratory, Texas A&M University) for amino acid analysis.


  • Ca-ATPase
  • EPR
  • calcium transport
  • cardiac
  • regulation


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