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Published on: July 4, 2016
Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy
Mark D Rustad1, Osha Roopnarine2, Razvan L Cornea2
1Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN, 55455, USA; School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, 55455, USA.
Insights
The novel DWORF micropeptide competes with Phospholamban (PLB) to regulate sarco/endoplasmic reticulum calcium ATPase (SERCA) activity. This interaction is crucial for understanding and potentially treating heart failure by improving calcium handling in cardiac muscle.
Area of Science:
- Biochemistry
- Molecular Biology
- Cardiovascular Research
Background:
- Sarco/endoplasmic reticulum calcium ATPase (SERCA) dysfunction is a key factor in heart failure, leading to impaired cardiac calcium (Ca2+) handling.
- Phospholamban (PLB) allosterically inhibits SERCA, reducing its efficiency in removing Ca2+ from the cytoplasm during diastole.
- The recently discovered dwarf open reading frame (DWORF) micropeptide acts as a muscle-specific effector of SERCA, counteracting PLB inhibition and activating SERCA independently.
Purpose of the Study:
- To elucidate the structural basis of DWORF's interaction with SERCA and PLB.
- To investigate the competitive binding dynamics between DWORF and PLB on SERCA.
- To refine molecular models of SERCA regulation for potential therapeutic applications in cardiac pathologies.
Main Methods:
- Co-reconstitution of SERCA, PLB, and DWORF in proteoliposomes.
- Utilizing electron paramagnetic resonance (EPR) spectroscopy with spin-labeled PLB.
- Analyzing changes in PLB rotational mobility to quantify competitive binding interactions.
Main Results:
- DWORF competes with PLB for binding to SERCA, particularly at low Ca2+ concentrations.
- DWORF exhibits a weaker binding affinity for SERCA compared to PLB, suggesting a cooperative regulatory mechanism.
- EPR spectroscopy provided insights into the dynamic structural changes governing SERCA regulation.
Conclusions:
- DWORF modulates SERCA activity through competitive binding with PLB.
- Understanding these protein-protein interactions offers a novel avenue for developing therapeutic strategies for heart failure.
- Further refinement of the molecular model for SERCA regulation is essential for advancing cardiac disease treatments.
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.
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