Gofman Yana, Schärfe Charlotta, Marks Debora S, Haliloglu Turkan, Ben-Tal Nir
Department of Biochemistry and Molecular Biology, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Tel Aviv, Israel.
Center for Bioinformatics, Quantitative Biology Center, and Department of Computer Science, Tübingen University, Tübingen, Germany; Department of Systems Biology, Harvard University, Boston, Massachusetts, United States of America.
PLoS Comput Biol. 2014 Dec 4;10(12):e1003976. doi: 10.1371/journal.pcbi.1003976. eCollection 2014 Dec.
Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels, essential for visual and olfactory sensory transduction. Although the channels include voltage-sensor domains (VSDs), their conductance is thought to be independent of the membrane potential, and their gating regulated by cytosolic cyclic nucleotide-binding domains. Mutations in these channels result in severe, degenerative retinal diseases, which remain untreatable. The lack of structural information on CNG channels has prevented mechanistic understanding of disease-causing mutations, precluded structure-based drug design, and hampered in silico investigation of the gating mechanism. To address this, we built a 3D model of the cone tetrameric CNG channel, based on homology to two distinct templates with known structures: the transmembrane (TM) domain of a bacterial channel, and the cyclic nucleotide-binding domain of the mouse HCN2 channel. Since the TM-domain template had low sequence-similarity to the TM domains of the CNG channels, and to reconcile conflicts between the two templates, we developed a novel, hybrid approach, combining homology modeling with evolutionary coupling constraints. Next, we used elastic network analysis of the model structure to investigate global motions of the channel and to elucidate its gating mechanism. We found the following: (i) In the main mode of motion, the TM and cytosolic domains counter-rotated around the membrane normal. We related this motion to gating, a proposition that is supported by previous experimental data, and by comparison to the known gating mechanism of the bacterial KirBac channel. (ii) The VSDs could facilitate gating (supplementing the pore gate), explaining their presence in such 'voltage-insensitive' channels. (iii) Our elastic network model analysis of the CNGA3 channel supports a modular model of allosteric gating, according to which protein domains are quasi-independent: they can move independently, but are coupled to each other allosterically.
环核苷酸门控（CNG）离子通道是一种非选择性阳离子通道，对视觉和嗅觉感觉转导至关重要。尽管这些通道包含电压传感器结构域（VSD），但其电导被认为与膜电位无关，其门控由胞质环核苷酸结合结构域调节。这些通道中的突变会导致严重的退行性视网膜疾病，目前仍无法治疗。由于缺乏关于CNG通道的结构信息，阻碍了对致病突变的机制理解，排除了基于结构的药物设计，并妨碍了对门控机制的计算机模拟研究。为了解决这个问题，我们基于与两个具有已知结构的不同模板的同源性，构建了锥体细胞四聚体CNG通道的三维模型：一个细菌通道的跨膜（TM）结构域和小鼠HCN2通道的环核苷酸结合结构域。由于TM结构域模板与CNG通道的TM结构域序列相似性较低，为了协调两个模板之间的冲突，我们开发了一种新颖的混合方法，将同源建模与进化耦合约束相结合。接下来，我们使用模型结构的弹性网络分析来研究通道的整体运动，并阐明其门控机制。我们发现了以下几点：（i）在主要运动模式中，TM结构域和胞质结构域围绕膜法线反向旋转。我们将这种运动与门控联系起来，这一观点得到了先前实验数据的支持，并与细菌KirBac通道的已知门控机制进行了比较。（ii）VSD可以促进门控（补充孔道门控），解释了它们在这种“电压不敏感”通道中的存在。（iii）我们对CNGA3通道的弹性网络模型分析支持变构门控的模块化模型，根据该模型，蛋白质结构域几乎是独立的：它们可以独立移动，但通过变构相互耦合。
