title: Protein-protein interactions in calcium transport regulation probed by saturation transfer electron paramagnetic resonance.
abstract: We have used electron paramagnetic resonance (EPR) to probe the homo- and heterooligomeric interactions of reconstituted sarcoplasmic reticulum Ca-ATPase (SERCA) and its regulator phospholamban (PLB). SERCA is responsible for restoring calcium to the sarcoplasmic reticulum to allow muscle relaxation, whereas PLB inhibits cardiac SERCA unless phosphorylated at Ser(16). To determine whether changes in protein association play essential roles in regulation, we detected the microsecond rotational diffusion of both proteins using saturation transfer EPR. Peptide synthesis was used to create a fully functional and monomeric PLB mutant with a spin label rigidly coupled to the backbone of the transmembrane helix, while SERCA was reacted with a Cys-specific spin label. Saturation transfer EPR revealed that sufficiently high lipid/protein ratios minimized self-association for both proteins. Under these dilute conditions, labeled PLB was substantially immobilized after co-reconstitution with unlabeled SERCA, reflecting their association to form the regulatory complex. Ser(16) phosphorylation slightly increased this immobilization. Complementary measurements with labeled SERCA showed no change in mobility after co-reconstitution with unlabeled PLB, regardless of its phosphorylation state. We conclude that phosphorylating monomeric PLB can relieve SERCA inhibition without changes in the oligomeric states of these proteins, indicating a structural rearrangement within the heterodimeric regulatory complex.
authors.affiliation: Department of Biochemistry, University of Minnesota, Minneapolis, Minnesota, USA.
authors.forename: Zachary M
authors.initials: ZM
authors.lastname: James
authors.forename: Jesse E
authors.initials: JE
authors.lastname: McCaffrey
authors.forename: Kurt D
authors.initials: KD
authors.lastname: Torgersen
authors.forename: Christine B
authors.initials: CB
authors.lastname: Karim
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D012399:Rotation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D013113:Spin Labels; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D013113:Spin Labels; D000099211:Phospholamban