Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers

Deo R. Singh, Michael P. Dalton, Ellen E. Cho, Marsha P. Pribadi, Taylor J. Zak, Jaroslava Šeflová, Catherine A. Makarewich, Eric N Olson, Seth L. Robia

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Abstract

The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA. In view of these two alternative binding interactions, we evaluated the possibility that oligomerization occurs at the expense of SERCA-binding. However, even the most avidly oligomeric micropeptide species still showed robust FRET with SERCA, and there was a surprising positive correlation between oligomerization affinity and SERCA-binding. This comparison of micropeptide family members suggests that the same structural determinants that support oligomerization are also important for binding to SERCA. Moreover, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory function of the other SERCA-binding micropeptides.

Original languageEnglish (US)
JournalJournal of Molecular Biology
DOIs
Publication statusAccepted/In press - Jan 1 2019

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Keywords

  • Calcium signaling
  • fluorescence resonance energy transfer
  • ion transport
  • Oligomerization
  • Protein–protein interactions

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Singh, D. R., Dalton, M. P., Cho, E. E., Pribadi, M. P., Zak, T. J., Šeflová, J., ... Robia, S. L. (Accepted/In press). Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers. Journal of Molecular Biology. https://doi.org/10.1016/j.jmb.2019.07.037