Asahi Michio, Sugita Yuji, Kurzydlowski Kazimierz, De Leon Stella, Tada Michihiko, Toyoshima Chikashi, MacLennan David H
Banting and Best Department of Medical Research, University of Toronto, Toronto, ON, Canada M5G 1L6.
Proc Natl Acad Sci U S A. 2003 Apr 29;100(9):5040-5. doi: 10.1073/pnas.0330962100. Epub 2003 Apr 11.
Phospholamban (PLN), a regulator of sarco(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs), interacts with both the cytosolic N domain and transmembrane helices M2, M4, M6, and M9 of SERCA. Amino acids in the transmembrane domain of PLN that are predicted to interact with SERCA1a are conserved in sarcolipin (SLN), a functional PLN homologue. Accordingly, the effects of critical mutations in SERCA1a, PLN, and NF-SLN (SLN tagged N-terminally with a FLAG epitope) on NF-SLN/SERCA1a and PLN/NF-SLN/SERCA1a interactions were compared. Critical mutations in SERCA1a and NF-SLN diminished functional interactions between SERCA1a and NF-SLN, indicating that NF-SLN and PLN interact with some of the same amino acids in SERCA1a. Mutations in PLN or NF-SLN affected the amount of SERCA1a that was coimmunoprecipitated in each complex with antibodies against either PLN or SLN, but not the pattern of coimmunoprecipitation. PLN mutations had more dramatic effects on SERCA1a coimmunoprecipitation than SLN mutations, suggesting that PLN dominates in the primary interaction with SERCA1a. Coimmunoprecipitation also confirmed that PLN and NF-SLN form a heterodimer that interacts with SERCA1a in a regulatory fashion to form a very stable PLN/NF-SLN/SERCA1a complex. Modeling showed that the SLN/SERCA1a complex closely resembles the PLN/SERCA1a complex, but with the luminal end of SLN extending to the loop connecting M1 and M2, where Tyr-29 and Tyr-31 interact with aromatic residues in SERCA1a. Modeling of the PLN/SLN/SERCA1a complex predicts that the regulator binding cavity in the E(2) conformation of SERCA1a can accommodate both SLN and PLN helices, but not two PLN helices.
受磷蛋白（PLN）是肌浆网钙-ATP酶（SERCAs）的一种调节因子，它与SERCA的胞质N结构域以及跨膜螺旋M2、M4、M6和M9相互作用。PLN跨膜结构域中预测与SERCA1a相互作用的氨基酸在肌脂蛋白（SLN）中是保守的，SLN是一种功能性PLN同源物。因此，比较了SERCA1a、PLN和NF-SLN（N端带有FLAG表位标签的SLN）中关键突变对NF-SLN/SERCA1a和PLN/NF-SLN/SERCA1a相互作用的影响。SERCA1a和NF-SLN中的关键突变减少了SERCA1a与NF-SLN之间的功能相互作用，表明NF-SLN和PLN与SERCA1a中的一些相同氨基酸相互作用。PLN或NF-SLN中的突变影响了与抗PLN或SLN抗体在每种复合物中共免疫沉淀的SERCA1a的量，但不影响共免疫沉淀模式。PLN突变对SERCA1a共免疫沉淀的影响比SLN突变更显著，表明PLN在与SERCA1a的主要相互作用中起主导作用。共免疫沉淀还证实，PLN和NF-SLN形成异二聚体，以调节方式与SERCA1a相互作用，形成非常稳定的PLN/NF-SLN/SERCA1a复合物。模型显示，SLN/SERCA1a复合物与PLN/SERCA1a复合物非常相似，但SLN的腔端延伸至连接M1和M2的环，其中Tyr-29和Tyr-31与SERCA1a中的芳香族残基相互作用。PLN/SLN/SERCA1a复合物的模型预测，SERCA1a的E(2)构象中的调节因子结合腔可以容纳SLN和PLN螺旋，但不能容纳两个PLN螺旋。
