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

• Published in: bioRxiv
• Main Authors: Ang, Li, Stroik, Daniel R, Yuen, Samantha L, Kleinboehl, Evan, Cornea, Razvan L, Thomas, David D
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 21.09.2020
• Subjects: Binding sites; Biosensors; Ca2+-transporting ATPase; Calcium (intracellular); Calcium (reticular); Calcium influx; Calcium transport; Cardiac muscle; Cardiomyocytes; Congestive heart failure; Developmental stages; Fluorescence resonance energy transfer; Green fluorescent protein; Heart failure; Membrane proteins; Muscle contraction; Myocytes; Phospholamban; Protein interaction; Proteins; Red fluorescent protein; Sarcoplasmic reticulum; Site-directed mutagenesis; Transmembrane domains
• DOI: 10.1101/2020.09.18.303644

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.