Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy

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Abstract

Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.

Original languageEnglish (US)
Pages (from-to)97-102
Number of pages6
JournalBiochemical and Biophysical Research Communications
Volume645
DOIs
StatePublished - Feb 19 2023

Bibliographical note

Funding Information:
This work was supported in part by NIH grants to DDT ( R01GM27906 and R37AG026160 ) and to DDT and RLC ( MPI , R01HL139065 ). MDR was funded by NIH training grant T32AR07612 . EPR experiments were performed at the Biophysical Technology Center, University of Minnesota. Dr. Ang Li and Dr. Peter D. Martin contributed to the early phase of this study. We thank Dr. Daniel Stroik and Dr. J. Michael Autry for helpful discussions.

Publisher Copyright:
© 2023 Elsevier Inc.

Keywords

  • DWORF
  • EPR
  • Membrane protein dynamics
  • Phospholamban
  • SERCA
  • Spin label

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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