Rearrangement of domain elements of the Ca-ATPase in cardiac sarcoplasmic reticulum membranes upon phospholamban phosphorylation.
Phospholamban (PLB) is a major target of the beta-adrenergic cascade in the heart, and functions to modulate rate-limiting conformational transitions involving the transport activity of the Ca-ATPase. To investigate structural changes within the Ca-ATPase that result from the phosphorylation of PLB by cAMP-dependent protein kinase (PKA), we have covalently bound the long-lived phosphorescent probe erythrosin isothiocyanate (Er-ITC) to cytoplasmic sequences within the Ca-ATPase. Under these labeling conditions, the Ca-ATPase remains catalytically active, indicating that observed changes in rotational dynamics reflect normal conformational transitions. Two major Er-ITC labeling sites were identified using electrospray ionization mass spectrometry (ESI-MS), corresponding to Lys464 and Lys650, which are respectively located within the phosphorylation and nucleotide binding domains of the Ca-ATPase. Frequency-domain phosphorescence measurements of the rotational dynamics of Er-ITC bound to these cytoplasmic sequences within the Ca-ATPase permit the resolution of the dynamic structure of individual domain elements relative to the overall rotational motion of the entire Ca-ATPase polypeptide chain. We observe a significant decrease in the rotational dynamics of Er-ITC bound to the Ca-ATPase upon phosphorylation of PLB by PKA, as evidenced by an increase in the residual anisotropy. These results suggest that phosphorylation of PLB results in a structural reorientation of the phosphorylation or nucleotide binding domains with respect to the membrane normal. In contrast, calcium activation of the Ca-ATPase in the presence of dephosphorylated PLB results in no detectable change in the rotational dynamics of Er-ITC, suggesting that calcium binding and PLB phosphorylation have distinct effects on the conformation of the Ca-ATPase. We suggest that PLB functions to alter the efficiency of phosphoenyzme formation following calcium activation of the Ca-ATPase by modulating the spatial arrangement between ATP bound in the nucleotide binding domain and Asp351 in the phosphorylation domain.
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Biochemistry and Biophysics Section, Department of Molecular Biosciences, University of Kansas, Lawrence 66045-2106, USA.
Huang
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Squier
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D000818:Animals; D016880:Anisotropy; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D004735:Energy Transfer; D004923:Erythrosine / Q000031:analogs & derivatives / Q000737:chemistry; D007425:Intracellular Membranes / Q000201:enzymology* / Q000378:metabolism; D017879:Isothiocyanates / Q000737:chemistry; D008163:Luminescent Measurements; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000201:enzymology* / Q000378:metabolism; D013113:Spin Labels; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004923:Erythrosine; D017879:Isothiocyanates; D010446:Peptide Fragments; D013113:Spin Labels; D000099211:Phospholamban; C022232:erythrosine isothiocyanate