Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base
Natronomonas pharaonis halorhodopsin Ser81 plays a role in maintaining chloride ions near the Schiff base
复制标题
Natronomonas pharaonis 盐视紫红质 Ser81 在维持席夫碱附近的氯离子方面发挥作用
DOI：
10.1016/j.bbrc.2018.06.156
复制
发表时间：
2018
影响因子：
3.1
通讯作者：
Tomita Hiroshi
中科院分区：
文献类型：
--
作者：
Sakajiri Yuko;Sugano Eriko;Watanabe Yoshito;Sakajiri Tetsuya;Tabata Kitako;Kikuchi Takeshi;Tomita Hiroshi
光遗传学技术常被用作光激活或沉默神经元的工具，嗜盐菌嗜盐视紫红质（NpHR）是一种光驱动氯离子泵。在光吸收时，氯离子穿过细胞膜，这伴随着氯离子与NpHR中质子化席夫碱附近的结合位点-1（BS 1）处的Thr 126的暂时结合。然而，还没有研究氯离子和BS 1之间的结合状态的稳定机制。因此，为了确定氯离子转运途径的关键组成部分，以及获取有关氯离子-BS 1结合状态的动态信息，我们对氯离子途径形状进行了粗略分析，然后对野生型和突变型NpHR结构进行了分子动力学（MD）模拟。分子动力学模拟表明，野生型蛋白中Thr 126与氯离子之间的氢键得以保留，而S81 A突变体中氯离子不能保留在BS 1处，并倾向于离开BS 1。我们发现，Thr 126侧链的方向是固定的Ser 81的羟基通过氢键和Thr 126绑定到一个氯离子在野生型蛋白质，而这种相互作用在S81 A突变体中丢失，导致旋转的Thr 126侧链和减少Thr 126和氯离子之间的相互作用。为了证实S81的作用，使用表达NpHR S81 A突变蛋白的细胞进行膜片钳记录。结合NpHR S81 A表达细胞在光刺激下不发生超极化的结果，我们的结果表明Ser 81在氯离子迁移中起关键作用。我们的发现可能与正在进行的使用光遗传基因疗法治疗盲人患者的临床试验有关。
Optogenetic technologies have often been used as tools for neuronal activation or silencing by light.Natronomonas pharaonishalorhodopsin (NpHR) is a light-driven chloride ion pump. Upon light absorption, a chloride ion passes through the cell membrane, which is accompanied by the temporary binding of a chloride ion with Thr126 at binding site-1 (BS1) near the protonated Schiff base in NpHR. However, the mechanism of stabilization of the binding state between a chloride ion and BS1 has not been investigated. Therefore, to identify a key component of the chloride ion transport pathway as well as to acquire dynamic information about the chloride ion-BS1 binding state, we performed a rough analysis of the chloride ion pathway shape followed by molecular dynamics (MD) simulations for both wild-type and mutant NpHR structures. The MD simulations showed that the hydrogen bond between Thr126 and the chloride ion was retained in the wild-type protein, while the chloride ion could not be retained at and tended to leave BS1 in the S81A mutant. We found that the direction of the Thr126 side chain was fixed by a hydroxyl group of Ser81 through a hydrogen bond and that Thr126 bound to a chloride ion in the wild-type protein, while this interaction was lost in the S81A mutant, resulting in rotation of the Thr126 side chain and reduction in the interaction between Thr126 and a chloride ion. To confirm the role of S81, patch clamp recordings were performed using cells expressing NpHR S81A mutant protein. Considered together with the results that the NpHR S81A-expressing cells did not undergo hyperpolarization under light stimulation, our results indicate that Ser81 plays a key role in chloride migration. Our findings might be relevant to ongoing clinical trials using optogenetic gene therapy in blind patients.