scholarly journals Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

2021 ◽  
Author(s):  
Sean R. Cleary ◽  
Xuan Fang ◽  
Ellen E. Cho ◽  
Marsha P. Pribadi ◽  
Jaroslava Seflova ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Ion Pump ◽  
Competing Interactions ◽  
Relaxation Cycle ◽  
Reading Frame ◽  
Preferential Binding ◽  
Physical Interactions ◽  
Mechanistic Basis ◽  
Dynamic Exchange

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the heart's adaptation to the increased physiological demands of exercise.

scholarly journals Aurora B dynamics at centromeres create a diffusion-based phosphorylation gradient

2011 ◽  
Vol 194 (4) ◽  
pp. 539-549 ◽  
Author(s):  
Enxiu Wang ◽  
Edward R. Ballister ◽  
Michael A. Lampson
Keyword(s):  
Cell Division ◽  
Spatial Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Aurora B ◽  
Kinase Activation ◽  
Chromosomal Site ◽  
Substrate Phosphorylation ◽  
Dynamic Exchange ◽  
And Diffusion

Aurora B kinase is essential for successful cell division and regulates spindle assembly and kinetochore–microtubule interactions. The kinase localizes to the inner centromere until anaphase, but many of its substrates have distinct localizations, for example on chromosome arms and at kinetochores. Furthermore, substrate phosphorylation depends on distance from the kinase. How the kinase reaches substrates at a distance and how spatial phosphorylation patterns are determined are unknown. In this paper, we show that a phosphorylation gradient is produced by Aurora B concentration and activation at centromeres and release and diffusion to reach substrates at a distance. Kinase concentration, either at centromeres or at another chromosomal site, is necessary for activity globally. By experimentally manipulating dynamic exchange at centromeres, we demonstrate that the kinase reaches its substrates by diffusion. We also directly observe, using a fluorescence resonance energy transfer–based biosensor, phosphorylation spreading from centromeres after kinase activation. We propose that Aurora B dynamics and diffusion from the inner centromere create spatial information to regulate cell division.

scholarly journals Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
M'Lynn E Fisher ◽  
Elisa Bovo ◽  
Rodrigo Aguayo-Ortiz ◽  
Ellen E Cho ◽  
Marsha P Pribadi ◽  
...  
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Critical Role ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cytosolic Calcium ◽  
Open Reading Frame ◽  
Calcium Pump ◽  
Reading Frame ◽  
Sarcoplasmic Reticulum Calcium ◽  
Direct Activator

The sarcoplasmic reticulum calcium pump SERCA plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

Quantum Dot Bioconjugates as Energy Donors in Fluorescence Resonance Energy Transfer Assays

2003 ◽  
Vol 773 ◽  
Author(s):  
Aaron R. Clapp ◽  
Igor L. Medintz ◽  
J. Matthew Mauro ◽  
Hedi Mattoussi
Keyword(s):  
Energy Transfer ◽  
Quantum Dot ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Molar Ratio ◽  
Binding Assays ◽  
Specific Sequence ◽  
Donor Acceptor

AbstractLuminescent CdSe-ZnS core-shell quantum dot (QD) bioconjugates were used as energy donors in fluorescent resonance energy transfer (FRET) binding assays. The QDs were coated with saturating amounts of genetically engineered maltose binding protein (MBP) using a noncovalent immobilization process, and Cy3 organic dyes covalently attached at a specific sequence to MBP were used as energy acceptor molecules. Energy transfer efficiency was measured as a function of the MBP-Cy3/QD molar ratio for two different donor fluorescence emissions (different QD core sizes). Apparent donor-acceptor distances were determined from these FRET studies, and the measured distances are consistent with QD-protein conjugate dimensions previously determined from structural studies.

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