钙库控制钙离子通道的调控机制的分子模拟研究

Calcium release-activated calcium (CRAC) channels are integral proteins of the plasma membrane that play a central role in cellular signaling. How calcium ions pass through the central pore of a CRAC channel (CRAC) is a key question underlying cellular communication. CRAC have two important components, the transmembrane pore subunit Orai and the calcium sensor STIM which is also the activator of Orai. Given the extensive biochemical and electrophysiological studies on CRAC channel, its gating mechanism remains elusive. Preliminary studies from our group have shown that an intrinsic property of the channel play a key role in determining the conductance state of Orai. However, it is unknown how STIM specifically functions to activate the pore, and the delicate balance of cooperative contributions from internal and external factors is poorly understood. Multiscale molecular modelling provides a robust computational tool to probe ion channel systems on microsecond timescales. It may be used to explore in detail the conformational change of the channel, yielding predictions in good agreement with available experimental data. In previous work, we have used computer simulations to study some important membrane proteins, including M2 proton channel, glutamate receptor, P2X4 channel as well as CRAC channel, and have gained experience to explore the structure and function of such complicated systems. Based on our preliminary work, we propose a research plan to address the mechanism of CRAC channel gating using large-scaling molecular simulations. Thus, the following Specific Aims are proposed: 1) study the mechanism of constitutive pore activation in the G98P, H134A and P245A Orai mutant structures, to understand the contributions to channel gating from intrinsic properties, such as kinking of pore-lining helices; 2) study the structure of STIM, and identify the possible conformation at the activated state, which could bind Orai to open the channel; 3) build a structure for the wild-type Orai-STIM complex, and develop a working model for STIM-triggered CRAC activation. Based on these results, comprehensive comparisons will be made (e.g., between different intrinsic factors towards channel gating, and between channel gating with and without STIM), which will lead to a better understanding of this ubiquitous and vital Ca2+ entry pathway.

钙库控制钙离子（CRAC）通道是控制钙离子进出细胞的重要膜蛋白。其有两个主要的组成部分，一个是跨膜的孔道蛋白Orai，一个是感受钙离子浓度变化的STIM蛋白，同时也是调控Orai孔道的激活蛋白。尽管文献中有一些关于CRAC通道的生化和电生理实验结果报道，钙通道的调控机制至今并不清晰。近年来，多尺度模拟的手段被广泛的用于研究复杂体系，例如研究离子通道蛋白的结构变化等性质并得到了与实验观测相吻合的结果。在以往的工作中，申请人采用多尺度模拟的手段研究了包括CRAC通道在内的数个重要的膜蛋白体系，积累了丰富的经验。在前期工作的基础上，我们拟从以下三个方面进一步开展系统研究，具体包括：（1）孔道蛋白Orai的不同功能状态；（2）激活蛋白STIM不同的功能状态；（3）Orai-STIM复合物的构建和调控机制。通过开展对于以上三个紧密联系又相互独立的课题的研究，我们有望充分理解钙离子进入细胞的分子机制。

钙库控制钙离子通道（CRAC）是调控钙离子进出细胞的重要跨膜蛋白，对钙信号传导具有作用。跨膜的孔道蛋白Orai和激活蛋白STIM是该通道的两个组成部分。尽管文献中有CRAC通道的生化和电生理实验结果报道，但其信号传导机制至今并不清晰。本项目中，我们采用多尺度模拟的手段，研究了CRAC通道的激活和调控的分子机制，在如下四个方面取得了进展：（1）我们发展了一种基于特征空间的构象采样方法DA2/teDA2。该方法对于局部构象空间，采用多条平行短轨迹进行充分采样，并采用数据降维对原子轨迹的协方差矩阵进行特征分解，找出占据主导地位的若干重要的运动模式以构成低维的特征空间，从而在自适应更新的特征空间中有效驱动结构变化。本方法对于CRAC通道的构象变化、调控及信号传导机制提供了重要的理论工具。（2）我们采用基于分子模拟的构象采样，结合自由能计算、有限元分析等理论手段，提出了Orai蛋白处于“开放态”的理论结构模型。基于该模型，可以得出孔道蛋白Orai的激活过程采用“twist-to-open”的门控机制。我们进一步与实验课题组合作，通过荧光成像和电生理实验证实了上述结构模型的合理性。（3）我们系统研究了Orai的若干具有代表性的突变体。结果表明，无论是结构变化、离子通过的自由能、还是电生理性质，突变体与野生型通道均有显著的区别。因此我们认为，针对突变体的研究得到的信息，均只能部分的但无法全面的反映野生型通道的激活和调控机制；对野生型通道蛋白质结构变化与信号传导之间的耦合关系是深入理解钙离子实施生理学功能的必要环节。（4）我们系统分析了STIM蛋白活化的分子机制。采用蛋白质工程方法结合分子模拟，我们确定了在生理条件下STIM的钙亲和力以及关键的氨基酸，并探讨了其对于STIM动力学过程的调控。上述工作，顺利完成了本项目既定的目标，对于从分子层面理解钙离子进入细胞的机制提供了重要的信息。