title: Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.
abstract: The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.
authors.affiliation: Department of Cell and Molecular Physiology, Loyola University Chicago, Maywood, Illinois.
authors.forename: Sandeep
authors.initials: S
authors.lastname: Pallikkuth
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Blackwell
authors.forename: Zhihong
authors.initials: Z
authors.lastname: Hu
authors.forename: Zhanjia
authors.initials: Z
authors.lastname: Hou
authors.forename: Dane T
authors.initials: DT
authors.lastname: Zieman
authors.forename: Bengt
authors.initials: B
authors.lastname: Svensson
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.forename: Seth L
authors.initials: SL
authors.lastname: Robia
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D031541:Fluorescence Resonance Energy Transfer; D008958:Models, Molecular; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D032383:Myocytes, Cardiac / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011487:Protein Conformation; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility; D014162:Transfection; D000099211:Phospholamban
chemical_list: D002135:Calcium-Binding Proteins; D003902:Detergents; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban