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Tracking Ca2+ ATPase intermediates in real time by x-ray solution scattering
Science Advances ( IF 11.7 ) Pub Date : 2020-03-20 , DOI: 10.1126/sciadv.aaz0981
Harsha Ravishankar 1 , Martin Nors Pedersen 2 , Mattias Eklund , Aljona Sitsel 3 , Chenge Li 4 , Annette Duelli 5 , Matteo Levantino 2, 6 , Michael Wulff 2 , Andreas Barth 4 , Claus Olesen 7 , Poul Nissen 3 , Magnus Andersson 1
Affiliation  

Sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) transporters regulate calcium signaling by active calcium ion reuptake to internal stores. Structural transitions associated with transport have been characterized by x-ray crystallography, but critical intermediates involved in the accessibility switch across the membrane are missing. We combined time-resolved x-ray solution scattering (TR-XSS) experiments and molecular dynamics (MD) simulations for real-time tracking of concerted SERCA reaction cycle dynamics in the native membrane. The equilibrium [Ca2]E1 state before laser activation differed in the domain arrangement compared with crystal structures, and following laser-induced release of caged ATP, a 1.5-ms intermediate was formed that showed closure of the cytoplasmic domains typical of E1 states with bound Ca2+ and ATP. A subsequent 13-ms transient state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. Hence, the obtained TR-XSS models determine the relative timing of so-far elusive domain rearrangements in a native environment.



中文翻译:

通过X射线溶液散射实时跟踪Ca2 + ATPase中间体

sarco /内质网Ca 2+ ATPase(SERCA)转运蛋白通过主动将钙离子重新摄取到内部存储来调节钙信号传导。与运输有关的结构转变已通过X射线晶体学进行了表征,但缺少跨膜可及性转换所涉及的关键中间体。我们将时间分辨的X射线溶液散射(TR-XSS)实验和分子动力学(MD)模拟相结合,以实时跟踪天然膜中协调一致的SERCA反应循环动力学。平衡[Ca 2激光激活之前的] E1状态与晶体结构相比在结构域安排上有所不同,并且在激光诱导释放笼状ATP之后,形成了一个1.5-ms的中间体,显示出典型的具有结合的Ca 2+和ATP。随后的13毫秒瞬态显示了以前无法解析的促动器(A)域结构,该结构在磷酸化后暴露了ADP结合位点。因此,获得的TR-XSS模型确定了本机环境中迄今为止难以捉摸的域重排的相对时间。

更新日期:2020-03-21
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