SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation
SERCA mutant E309Q binds two Ca2+ ions but adopts a catalytically incompetent conformation
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DOI：
10.1038/emboj.2013.250
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发表时间：
2013-12-11
期刊：
影响因子：
11.4
通讯作者：
le Maire, Marc
中科院分区：
文献类型：
--
作者：
Clausen, Johannes D.;Bublitz, Maike;le Maire, Marc
肌浆网Ca ~（2+）-ATP酶（SERCA）是一种将ATP水解与Ca ~（2+）转运偶联的酶。这种定向能量转移需要两个Ca 2+位点和磷酸化位点之间超过50埃距离的串扰。我们已经解决了这个分子内信号的机械结构的基础上，通过分析SERCA突变体E309 Q的结构和功能特性。Glu（309）有助于位点II处的Ca 2+配位，并且一致认为E309 Q仅在位点I处结合Ca 2+。然而，在Ca 2+和ATP类似物存在下，E309 Q的晶体结构揭示了非催化Ca（2）E1状态的两个占据的Ca 2+位点。Ca ~（2+）与E309 Q中的两个Ca ~（2+）位点以微摩尔亲和力结合，但没有协同性。Ca 2+结合突变体确实从ATP磷酸化，但最大速率非常低。磷酸化依赖于A结构域的正确定位，需要跨膜片段M1移位到“向上和扭结的位置”。这种转变是受损的E309 Q突变体，最有可能是由于缺乏电荷中和和改变的氢结合能力在Ca 2+网站II。
The sarco(endo) plasmic reticulum Ca2+-ATPase (SERCA) couples ATP hydrolysis to transport of Ca2+. This directed energy transfer requires cross-talk between the two Ca2+ sites and the phosphorylation site over 50 angstrom distance. We have addressed the mechano-structural basis for this intramolecular signal by analysing the structure and the functional properties of SERCA mutant E309Q. Glu(309) contributes to Ca2+ coordination at site II, and a consensus has been that E309Q only binds Ca2+ at site I. The crystal structure of E309Q in the presence of Ca2+ and an ATP analogue, however, reveals two occupied Ca2+ sites of a non-catalytic Ca(2)E1 state. Ca2+ is bound with micromolar affinity by both Ca2+ sites in E309Q, but without cooperativity. The Ca2+ -bound mutant does phosphorylate from ATP, but at a very low maximal rate. Phosphorylation depends on the correct positioning of the A-domain, requiring a shift of transmembrane segment M1 into an 'up and kinked position'. This transition is impaired in the E309Q mutant, most likely due to a lack of charge neutralization and altered hydrogen binding capacities at Ca2+ site II.