Phospholamban Modulates the Functional Coupling between Nucleotide Domains in Ca-ATPase Oligomeric Complexes in Cardiac Sarcoplasmic Reticulum†

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• 作者：Linda T. L. Chen^‡ ; ; Qing Yao^§ ; ; Thereza A. Soares ; Thomas C. Squier ; Diana J. Bigelow*
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：March 24, 2009
• 年：2009
• 卷：48
• 期：11
• 页码：2411-2421
• 全文大小：264K
• 年卷期：v.48,no.11(March 24, 2009)
• ISSN：1520-4995

文摘

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 Lys⁵¹⁴ 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 Cys⁶⁷⁴ 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 Å decrease in the proximity between FITC sites within the N-domain and a 9 ± 3 Å 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.