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bioRxiv · 10.1101/2020.09.18.303644

The transmembrane domain of DWORF activates SERCA directly; P15 and W22 residues are essential.

Li, A.·Stroik, D. R.·Yuen, S. L.·Kleinboehl, E.·Cornea, R. L.·Thomas, D. D.
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Abstract

The Ca-ATPase isoform 2a (SERCA2a) re-sequesters cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. A central factor in heart failure is abnormally high cytosolic [Ca2+], resulting in pathophysiology and decreased cardiac performance. Therefore, augmentation of the SERCA2a Ca2+ transport activity is a promising therapeutic approach. A novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing the protein phospholamban (PLB) from its inhibitory site on SERCA2a. In the present study, we have developed several cell-based FRET biosensor systems for time-resolved FRET (TR-FRET) measurements of the protein-protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the putative SERCA2a binding site, we transiently transfected DWORF into a stable cell line expressing SERCA2a labeled with green fluorescent protein (GFP, the FRET donor) and PLB labeled with red fluorescent protein (RFP, the FRET acceptor). We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Functional analysis demonstrates that DWORF activates SERCA in both the presence and absence of PLB. Furthermore, using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues that are necessary for functional SERCA2a-DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

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Li, A., Stroik, D. R., Yuen, S. L., Kleinboehl, E., Cornea, R. L., Thomas, D. D.. 2020-09-21. The transmembrane domain of DWORF activates SERCA directly; P15 and W22 residues are essential.. https://doi.org/10.1101/2020.09.18.303644

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