Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.
Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.
复制标题
DOI：
10.1016/j.jmb.2010.11.014
复制
发表时间：
2011-01-21
影响因子：
5.6
通讯作者：
Young HS
中科院分区：
文献类型：
--
作者：
Glaves JP;Trieber CA;Ceholski DK;Stokes DL;Young HS
磷蛋白与肌浆网钙泵（SERCA）相互作用，调节心脏对肾上腺素能刺激的收缩性。我们用电子显微镜研究了SERCA与磷蛋白复合物的二维晶体的相互作用。在先前的研究中，发现磷低聚物穿插在SERCA二聚体带之间，并构建了一个三维模型来显示与SERCA的相互作用。在本研究中，我们研究了磷蛋白的低聚状态，以及磷蛋白磷酸化和突变对二维晶体中SERCA相互作用的影响。根据阴性染色和冷冻水合晶体的投影图，Ser16的磷酸化选择性地扰乱了野生型磷蛋白的细胞质结构域。而对于一个五聚体功能获得突变体（Lys27-to-Ala）来说，情况并非如此，它保留了抑制活性，并保持了磷酸化状态。部分功能丧失突变改变了磷蛋白（Arg14-to-Ala）的电荷状态，保持了有序状态，而完全功能丧失突变（Asn34-to-Ala）也是无序的。磷蛋白的功能状态与二维共晶中磷蛋白胞质结构域的有序到无序转变有关。此外，功能获得突变体（Lys27-to-Ala）的共晶有助于从冷冻水合晶体中收集数据。改进的投影图计算到8 Å的分辨率，支持五聚体作为磷在晶体中的低聚态。与SERCA的二维共晶体需要一种功能性的五聚体形式的磷蛋白，它与SERCA在一个不同于磷蛋白单体用于抑制关联的辅助位点进行物理相互作用。
Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with phospholamban. In previous studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was also disordered. The functional state of phospholamban correlated with an order-to-disorder transition of phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27-to-Ala) facilitated data collection from frozen hydrated crystals. An improved projection map was calculated to a resolution of 8 Å, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The two-dimensional co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association.
登录
查看更多内容
影响因子：
4.8
作者：
Kimura, Y;Asahi, M;MacLennan, DH
通讯作者：
MacLennan, DH
影响因子：
3
作者：
Crowther, RA;Henderson, R;Smith, JM
通讯作者：
Smith, JM
影响因子：
64.8
作者：
Auer, M;Scarborough, GA;Kühlbrandt, W
通讯作者：
Kühlbrandt, W
影响因子：
4.8
作者：
Chu, GX;Li, L;Kranias, EG
通讯作者：
Kranias, EG
DOI：
10.1016/0005-2736(89)90323-4
发表时间：
1989-04-28
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
--
作者：
FOWLER, C;HUGGINS, JP;CHAPMAN, D
通讯作者：
CHAPMAN, D