Phospholamban modulates the functional coupling between nucleotide domains in Ca-ATPase oligomeric complexes in cardiac sarcoplasmic reticulum.
Oligomeric interactions between Ca-ATPase polypeptide chains and their modulation by phospholamban (PLB) were measured in native cardiac sarcoplasmic reticulum (SR) microsomes. Progressive modification of Lys(514) with fluorescein 5-isothiocyanate (FITC), which physically blocks access to the nucleotide binding site by ATP, demonstrates that Ca-ATPase active sites function independently of one another prior to the phosphorylation of PLB. However, upon cAMP-dependent protein kinase (PKA) phosphorylation of PLB, a second-order dependence between residual enzyme activity and the fraction of active sites is observed, consistent with a dimeric functional complex. Complementary distance measurements were made using FITC or 5-iodoacetamidofluorescein (IAF) bound to Cys(674) within the N- or P-domains, respectively, to detect structural coupling within oligomeric complexes. Accompanying the phosphorylation of PLB, neighboring Ca-ATPase polypeptide chains exhibit a 4 +/- 2 A decrease in the proximity between FITC sites within the N-domain and a 9 +/- 3 A increase in the proximity between IAF sites within P-domains. Thus, the phosphorylation of PLB induces spatial rearrangements between the N- and P-domain elements of proximal Ca-ATPase polypeptide chains which restore functional interactions between neighboring polypeptide chains and, in turn, result in increased rates of catalytic turnover. These results are interpreted in terms of a structural model, calculated through optimization of shape complementarity, desolvation, and electrostatic energies, which suggests a dimeric arrangement of Ca-ATPase polypeptide chains through the proximal association of N-domains that accommodates interaction with PLB. We suggest that the phosphorylation of PLB acts to release constraints involving interdomain subunit interactions that enhance catalytically important N-domain motions.
Chen
Linda T L
LT
Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Yao
Qing
Q
Soares
Thereza A
TA
Squier
Thomas C
TC
Bigelow
Diana J
DJ
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D002478:Cells, Cultured; D007700:Kinetics; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012519:Sarcoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D013552:Swine; D000099211:Phospholamban
D002118:Calcium; D002135:Calcium-Binding Proteins; D009711:Nucleotides; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban