Cytoplasmic interactions between phospholamban residues 1-20 and the calcium-activated ATPase of the sarcoplasmic reticulum.
Phospholamban regulates the activity of the calcium-activated ATPase (CaATPase) of cardiac sarcoplasmic reticulum. Equilibrium fluorescence studies have shown that the N-terminal cytoplasmic region of phospholamban (residues 1-20, domain 1) causes a decrease in the intrinsic tryptophan fluorescence of the CaATPase. The interaction of phospholamban residues 1-20 with the CaATPase also results in spectral changes for the extrinsic chromophore FITC covalently attached to the cytoplasmic region of the calcium pump. The fluorescence changes for both reporter groups correlate with a dissociation constant of approximately 40 microM for the complex between phospholamban residues 1-20 and the CaATPase. Complex formation is notably weaker when phospholamban 1-20 is titrated into the CaATPase in the presence of calcium, with altered conformational effects resulting from binding. The interaction of domain 1 of phospholamban with the CaATPase is also reduced upon phosphorylation of phospholamban 1-20 at Ser-16. This region of phospholamban 1-20 is shown by isotope-edited NMR study to be involved in interaction with the CaATPase. Binding of the phosphorylated peptide is not abolished, however, indicating that phospholamban 1-20 remains associated with the CaATPase even after phosphorylation. The data provide direct evidence for the interaction between the cytoplasmic regions of phospholamban and the pump, and are discussed in the context of the mechanism for inhibition of cardiac CaATPase activity by phospholamban.
Sharma
P
P
School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK.
Patchell
V B
VB
Gao
Y
Y
Evans
J S
JS
Levine
B A
BA
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D066298:In Vitro Techniques; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D009711:Nucleotides; D010446:Peptide Fragments; D000099211:Phospholamban