Biological Sciences Division, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352, USA.
Biochemistry. 2009 Mar 24;48(11):2411-21. doi: 10.1021/bi8021526.
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.
寡聚相互作用之间的 Ca-ATPase 多肽链和它们的调制磷酸化肌球蛋白轻链结合蛋白 (PLB) 进行了测量，在天然心脏肌浆网 (SR) 微粒体。逐步修改赖氨酸 (514) 与荧光素 5-异硫氰酸酯 (FITC)，这物理块访问核苷酸结合位点的 ATP，表明 Ca-ATPase 活性位点功能独立于另一个之前磷酸化 PLB。然而，在 cAMP 依赖性蛋白激酶 (PKA) 磷酸化 PLB，二阶之间的依赖关系残余酶活性和分数的活性位点是观察到的，与二聚体功能复合物。互补的距离测量是用 FITC 或 5-碘乙酰胺荧光素 (IAF) 绑定到 Cys(674) 内 N 或 P-域，分别检测结构耦合在寡聚体复合物。伴随着磷酸化 PLB，邻近的 Ca-ATPase 多肽链表现出 4 +/- 2 A 减少在接近 FITC 网站内 N-域和 9 +/- 3 A 增加在接近 IAF 网站内 P-域。因此，磷酸化 PLB 诱导空间重排之间的 N-和 P-域元素的邻近 Ca-ATPase 多肽链，恢复功能之间的相互作用邻近的多肽链，并依次导致增加的催化周转率。这些结果是解释结构模型，计算通过优化形状互补，去溶剂化，和静电能量，这表明二聚体排列的 Ca-ATPase 多肽链通过近端协会的 N-域，以适应与 PLB 的相互作用。我们认为，磷酸化 PLB 作用释放约束涉及的域间亚基相互作用，提高催化重要的 N-域运动。
