Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures.
Ligand discrimination and gating in cyclic nucleotide-gated ion channels from apo and partial agonist-bound cryo-EM structures.
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DOI：
10.7554/elife.39775
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发表时间：
2018-07-20
期刊：
影响因子：
7.7
通讯作者：
Nimigean CM
中科院分区：
文献类型：
--
作者：
Rheinberger J;Gao X;Schmidpeter PA;Nimigean CM
环核苷酸调节通道在视觉信号转导和起搏中起着重要作用。环核苷酸的结合（cAMP/cGMP）在家族内的不同通道中引起不同的功能反应，尽管它们具有高度的序列和结构同源性。由于结构信息和功能状态之间缺乏对应关系，负责配体识别和门控的分子机制尚不清楚。利用单粒子低温电子显微镜和单通道记录，我们确定了原核生物环核苷酸门控通道SthK的高分辨率结构的功能态。脂质纳米盘中载脂蛋白、camp结合和cgmp结合的SthK的结构分别对应于无、中等和低单通道活性，这与观察到的所有结构都处于静止、封闭状态一致。载脂蛋白和配体结合结构之间的相似性表明配体结合结构域以一种变构一致的方式与孔和shk门强耦合。cAMP和cGMP在“静止”和“激活”结构中的不同取向提示了一种配体识别机制。离子通道对于神经系统和大脑的信号传递至关重要。一大批离子通道包括被环状核苷酸激活的成员，环状核苷酸是用来在细胞内传递信号的小分子。这些环核苷酸门控通道在调节我们的视觉和嗅觉能力方面发挥着重要作用。这些离子通道的活性已经被研究了多年，但科学家们直到最近才能够观察到它们的结构。由于结构生物学方法需要纯化的、表现良好的蛋白质，因此选择用于结构研究的离子通道家族成员不一定与活性已经确定的离子通道家族成员相匹配。需要有一个好的模型，可以同时表征环核苷酸门控离子通道的结构和活性。来自嗜热螺旋体细菌的环核苷酸门控离子通道SthK被确定为这样的模型，因为它的活性和结构都是可接近的。Rheinberger等人利用低温电子显微镜解决了几个高分辨率的SthK通道结构。在其中两种结构中，SthK与两种激活环核苷酸（cAMP或cGMP）中的一种结合，而在另一种结构中，没有环核苷酸结合。单独记录单个通道的活动可以识别可能由这些结构表示的活动状态。结合实验结果发现，未结合状态下的通道没有活性，与cGMP结合的通道活性低，与cAMP结合的通道活性中等。Rheinberger等人表明，在低温电子显微镜所经历的条件下，通道在研究的所有状态下都是关闭的。出乎意料的是，即使在通道的环核苷酸结合口袋中，环核苷酸的结合也没有产生结构变化，而以前观察到，当该区域单独结晶时，该区域也会发生这种变化。Rheinberger等人由此推断，构成通道的四个亚基可能同时发生构象变化，向开放状态转变，而不是一个接一个。SthK通道的结构和基本功能特征为未来研究确定环核苷酸门控通道的整个开启和关闭周期提供了一个强有力的起点。人类等效的通道可能以类似的方式工作。因此，Rheinberger等人提出的结果可以帮助解决由环核苷酸门控通道缺陷引起的疾病，例如由于视网膜退化（色素性视网膜炎或进行性锥体营养不良）和色盲导致的视力丧失。
Cyclic nucleotide-modulated channels have important roles in visual signal transduction and pacemaking. Binding of cyclic nucleotides (cAMP/cGMP) elicits diverse functional responses in different channels within the family despite their high sequence and structure homology. The molecular mechanisms responsible for ligand discrimination and gating are unknown due to lack of correspondence between structural information and functional states. Using single particle cryo-electron microscopy and single-channel recording, we assigned functional states to high-resolution structures of SthK, a prokaryotic cyclic nucleotide-gated channel. The structures for apo, cAMP-bound, and cGMP-bound SthK in lipid nanodiscs, correspond to no, moderate, and low single-channel activity, respectively, consistent with the observation that all structures are in resting, closed states. The similarity between apo and ligand-bound structures indicates that ligand-binding domains are strongly coupled to pore and SthK gates in an allosteric, concerted fashion. The different orientations of cAMP and cGMP in the ‘resting’ and ‘activated’ structures suggest a mechanism for ligand discrimination. Ion channels are essential for transmitting signals in the nervous system and brain. One large group of ion channels includes members that are activated by cyclic nucleotides, small molecules used to transmit signals within cells. These cyclic nucleotide-gated channels play an important role in regulating our ability to see and smell. The activity of these ion channels has been studied for years, but scientists have only recently been able to look into their structure. Since structural biology methods require purified, well-behaved proteins, the members of this ion channel family selected for structural studies do not necessarily match those whose activity has been well established. There is a need for a good model that would allow both the structure and activity of a cyclic nucleotide-gated ion channel to be characterized. The cyclic nucleotide-gated ion channel, SthK, from bacteria called Spirochaeta thermophila, was identified as such model because both its activity and its structure are accessible. Rheinberger et al. have used cryo electron microscopy to solve several high-resolution structures of SthK channels. In two of the structures, SthK was bound to either one of two types of activating cyclic nucleotides – cAMP or cGMP – and in another structure, no cyclic nucleotides were bound. Separately recording the activity of individual channels allowed the activity states likely to be represented by these structures to be identified. Combining the results of the experiments revealed no activity from channels in an unbound state, low levels of activity for channels bound to cGMP, and moderate activity for channels bound to cAMP. Rheinberger et al. show that the channel, under the conditions experienced in cryo electron microscopy, is closed in all of the states studied. Unexpectedly, the binding of cyclic nucleotides produced no structural change even in the cyclic nucleotide-binding pocket of the channel, a region that was previously observed to undergo such changes when this region alone was crystallized. Rheinberger et al. deduce from this that the four subunits that make up the channel likely undergo the conformational change towards an open state all at once, rather than one by one. The structures and the basic functional characterization of SthK channels provide a strong starting point for future research into determining the entire opening and closing cycle for a cyclic nucleotide-gated channel. Human equivalents of the channel are likely to work in similar ways. The results presented by Rheinberger et al. could therefore be built upon to help address diseases that result from deficiencies in cyclic nucleotide-gated channels, such as loss of vision due to retinal degradation (retinitis pigmentosa or progressive cone dystrophy) and achromatopsia.