Zabelskii Dmitrii, Bukhdruker Sergey, Bukhalovich Siarhei, Tsybrov Fedor, Lamm Gerrit H U, Astashkin Roman, Doroginin Demid, Matveev Grigory, Sudarev Vsevolod, Kuzmin Alexander, Zinovev Egor, Vlasova Anastasiia, Ryzhykau Yury, Ilyinsky Nikolay, Gushchin Ivan, Bourenkov Gleb, Alekseev Alexey, Round Adam, Wachtveitl Josef, Bamberg Ernst, Gordeliy Valentin
European X-ray Free Electron Laser GmbH, Schenefeld, Germany.
Research Center for Molecular Mechanisms of Aging and Age-related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia.
Nat Struct Mol Biol. 2025 Apr 9. doi: 10.1038/s41594-025-01488-7.
Channelrhodopsins (ChRs) have emerged as major optogenetics tools, particularly in neuroscience. Despite their importance, the molecular mechanism of ChR opening remains elusive. Moreover, all reported structures of ChRs correspond to either a closed or an early intermediate state and lack the necessary level of detail owing to the limited resolution. Here we present the structures of the closed and open states of a cation-conducting ChR, OLPVR1, from Organic Lake phycodnavirus, belonging to the family of viral ChRs solved at 1.1- and 1.3-Å resolution at physiologically relevant pH conditions (pH 8.0). OLPVR1 was expressed in Escherichia coli and crystallized using an in meso approach, and the structures were solved by X-ray crystallography. We also present the structure of the OLPVR1 protonated state at acidic pH (pH 2.5) at 1.4-Å resolution. Together, these three structures elucidate the molecular mechanisms of the channel's opening and permeability in detail. Extensive functional studies support the proposed mechanisms. Channel opening is controlled by isomerization of the retinal cofactor, triggering protonation of proton acceptors and deprotonation of proton donors located in the three gates of the channel. The E51 residue in the core of the central gate (similar to E90 of ChR2 from Chlamydomonas reinhardtii) plays a key role in the opening of the channel. E51 flips out of the gate and towards the proton acceptor D200 (D253 in ChR2 in C. reinhardtii), establishing a hydrogen bond between them. Despite differences in subfamilies of ChRs, they share a common gate-cavity architecture, suggesting that they could have similar general gating mechanisms. These results enabled us to design viral rhodopsin with improved properties for optogenetic applications. The structural data and mechanisms might also be helpful for better understanding other ChRs and their engineering.
通道视紫红质（ChRs）已成为主要的光遗传学工具，尤其是在神经科学领域。尽管它们很重要，但ChR开放的分子机制仍然难以捉摸。此外，所有已报道的ChRs结构都对应于关闭状态或早期中间状态，并且由于分辨率有限而缺乏必要的细节水平。在这里，我们展示了一种来自有机湖藻DNA病毒的阳离子传导ChR——OLPVR1的关闭态和开放态结构，该病毒属于病毒ChRs家族，在生理相关pH条件（pH 8.0）下以1.1埃和1.3埃的分辨率解析得到。OLPVR1在大肠杆菌中表达，并采用中晶法结晶，结构通过X射线晶体学解析。我们还展示了OLPVR1在酸性pH（pH 2.5）下1.4埃分辨率的质子化状态结构。这三种结构共同详细阐明了通道开放和通透性的分子机制。广泛的功能研究支持了所提出的机制。通道开放由视网膜辅因子的异构化控制，触发质子受体的质子化和位于通道三个门控处的质子供体的去质子化。中央门控核心中的E51残基（类似于莱茵衣藻ChR2中的E90）在通道开放中起关键作用。E51从门控处翻转出来并朝向质子受体D200（莱茵衣藻ChR2中的D253），在它们之间形成氢键。尽管ChRs亚家族存在差异，但它们共享共同的门控-腔结构，这表明它们可能具有相似的一般门控机制。这些结果使我们能够设计出具有改进特性的病毒视紫红质用于光遗传学应用。这些结构数据和机制也可能有助于更好地理解其他ChRs及其工程改造。
