Mapping the interaction surface of a membrane protein: Unveiling the conformational switch of phospholamban in calcium pump regulation

J. Zamoon, F. Nitu, C. Karim, D. D. Thomas, G. Veglia

Research output: Contribution to journalArticle

92 Scopus citations

Abstract

We have used magnetic resonance to map the interaction surface of an integral membrane protein for its regulatory target, an integral membrane enzyme. Phospholamban (PLN) regulates cardiac contractility via its modulation of sarco(endo)plasmic reticulum calcium ATPase (SERCA) activity. Impairment of this regulatory process causes heart failure. To map the molecular details of the PLN/SERCA interaction, we have functionally reconstituted SERCA with labeled PLN in dodecylphosphocholine micelles for high-resolution NMR spectroscopy and in both micelles and lipid bilayers for EPR spectroscopy. Differential perturbations in NMR linewidths and chemical shifts, measured as a function of position in the PLN sequence, provide a vivid picture of extensive SERCA contacts in both cytoplasmic and transmembrane domains of PLN and provide structural insight into previously reported functional mutagenesis data. NMR and EPR data show clear and complementary evidence for a dynamic (μs-to-ms) equilibrium between two conformational states in the cytoplasmic domain of PLN. These results support the hypothesis that SERCA attracts the cytoplasmic domain of PLN away from the lipid surface, shifting the preexisting equilibrium of PLN conformers toward a structure that is poised to interact with the regulatory target. EPR shows that this conformational switch behaves similarly in micelles and lipid membranes. Based on structural and dynamics data, we propose a model in which PLN undergoes allosteric activation upon encountering SERCA.

Original languageEnglish (US)
Pages (from-to)4747-4752
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number13
DOIs
StatePublished - Mar 29 2005

Keywords

  • NMR spectroscopy
  • Protein-protein interaction

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