Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION
Formation of the Stable Structural Analog of ADP-sensitive Phosphoenzyme of Ca2+-ATPase with Occluded Ca2+ by Beryllium Fluoride STRUCTURAL CHANGES DURING PHOSPHORYLATION AND ISOMERIZATION
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DOI：
10.1074/jbc.m109.029702
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发表时间：
2009-08-21
影响因子：
4.8
通讯作者：
Suzuki, Hiroshi
中科院分区：
文献类型：
--
作者：
Danko, Stefania;Daiho, Takashi;Suzuki, Hiroshi
作为肌浆网Ca ~（2+）-ATP酶ADP敏感磷酸化中间体的稳定类似物，E_1Ca（2）中心点Mg通过在Ca ~（2+）结合态E_1Ca（2）上加入氟化铍和Mg ~（2+）而形成了E_1Ca（2）中心点BeFx的复合物。在E1 Ca（2）中心点BeFx中，最可能是E1 Ca（2）中心点BeF 3-，两个Ca 2+被封闭在高亲和力转运位点，其形成需要Mg 2+在催化位点结合，ADP将其分解为E1 Ca（2），如在E1 PCa（2）中心点Mg中。E1 Ca（2）中心点BeFx中胞质结构域的组织被揭示为介于E1 Ca（2）中心点AlF 4- ADP（E1 PCa（2）形成的过渡态）和E2中心点BeF 3-（ADP不敏感的磷酸化中间体E2 P中心点Mg）中胞质结构域的组织之间。三硝基苯基-AMP（TNP-AMP）与E1 Ca（2）中心点BeFx形成强荧光（超荧光）复合物，而与E1 Ca（2）中心点AlFx结合的TNP-AMP无超荧光。E1 Ca（2）中心点BeFx与TNP-AMP结合后缓慢衰减为E1 Ca（2），与TNP-AMP与E2中心点BeF ~（3-）形成的超荧光复合物不同，后者稳定。色氨酸荧光分析表明，E1 Ca（2）中心点BeFx的跨膜结构与E1 PCa（2）中心点Mg相似，E1 Ca（2）中心点AlF 4-中心点ADP和E2中心点BeF 3-的跨膜结构介于两者之间。E1 Ca（2）中心点BeFx在低50-100 μ M Ca ~（2+）时缓慢转化为E2中心点BeF ~（3+）释放Ca ~（2+），模拟E1 PCa（2）中心点Mg ~+> E2 P中心点Mg ~+ 2Ca（2+）中心点Ca ~（2+）在约毫摩尔高Ca ~（2+）时催化位点上Mg ~（2+）的置换将E1 Ca（2）中心点BeFx分解为E1 Ca（2）。值得注意的是，E1 Ca（2）中心点BeFx被0.7 mM内腔Ca 2+和15 mM Mg 2+完全稳定至少12天。此外，通过将两个Ca 2+结合到管腔定向的低亲和力转运位点，在0.7mM管腔Ca 2+下从E2中心点BeF 3-产生稳定的E1 Ca（2）中心点BeFx，如模拟反向转化E2 P中心点Mg + 2Ca（2+）-> E1 PCa（2）中心点Mg。
As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase E1PCa(2)center dot Mg, a complex of E1Ca(2)center dot BeFx, was successfully developed by addition of beryllium fluoride and Mg2+ to the Ca2+-bound state, E1Ca(2). In E1Ca(2)center dot BeFx, most probably E1Ca(2)center dot BeF3-, two Ca2+ are occluded at high affinity transport sites, its formation required Mg2+ binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2)center dot Mg. Organization of cytoplasmic domains in E1Ca(2)center dot BeFx was revealed to be intermediate between those in E1Ca(2)center dot AlF4- ADP (transition state of E1PCa(2) formation) and E2 center dot BeF3-(ADP-insensitive phosphorylated intermediate E2P center dot Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2)center dot BeFx in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2)center dot AlFx. E1Ca(2)center dot BeFx with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2 center dot BeF3-, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2)center dot BeFx mimics E1PCa(2)center dot Mg, and between those of E1Ca(2)center dot AlF4-center dot ADP and E2 center dot BeF3-. E1Ca(2)center dot BeFx at low 50-100 mu M Ca2+ was converted slowly to E2 center dot BeF3- releasing Ca2+, mimicking E1PCa(2)center dot Mg -> E2P center dot Mg + 2Ca(2+)center dot Ca2+ replacement of Mg2+ at the catalytic site at approximately millimolar high Ca2+ decomposed E1Ca(2)center dot BeFx to E1Ca(2). Notably, E1Ca(2)center dot BeFx was perfectly stabilized for at least 12 days by 0.7 mM lumenal Ca2+ with 15 mM Mg2+. Also, stable E1Ca(2)center dot BeFx was produced from E2 center dot BeF3- at 0.7 mM lumenal Ca2+ by binding two Ca2+ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P center dot Mg + 2Ca(2+) -> E1PCa(2)center dot Mg.