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.

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。