Phytochromes: Structural Perspectives on Photoactivation and Signaling

项目总结 几乎所有的细胞生物都使用一组光感受器来检测它们的光环境。可以说是世界上 有影响力的是光敏色素(Phys)，这是一种对植物发育和许多细菌的行为至关重要的多样化群体， 真菌和藻类物种。通过可逆光相互转化它们的联苯(或开链四氢吡咯)发色团 在红光吸收Pr和远红光Pfr状态之间，物理在各种信号中充当光开关 级联响应光强度、持续时间、方向和光谱质量。此外，通过热还原， Pfr回到Pr，一些物理通过这个反应的热效应来感知温度，并通过光周期来感知 夜间PFR的耗尽。这种PR/PFR相互转换的累积效应影响了许多生理上的 对农业以及有害植物和人类病原体的生物学很重要的过程。此外，他们的独特之处 光化学提供了无价的光遗传工具，包括用于组织成像的新型荧光团，以及工程化的 用于调节细胞事件的光开关，具有显著的时间和空间精度。 最近，我们在理解Phys如何发出信号方面取得了很大进展，新出现的结构表明微生物和 植物物理使用两种不同的输出模式。两者都是从光触发的BLIN异构化开始的，这会驱动- 滞留到-连接签名PHY和GAF结构域的发夹环的螺旋重排。当被光激活时 然后，微生物物理连接在二聚体内产生的扭转应变以调节附加的输出域(通常 具有组氨酸激酶活性)，植物物理已经重新排列了它们的结构域组织以创建光敏二聚体 可能使PIF转录抑制物家族发生可逆结合并最终降解的平台。 虽然目前的模型有助于阐明终态转换所需的巨大变化，但光激发的中间产物 而创建具有信令能力的PFR状态所需的随之而来的结构性变化仍不确定。 这项计划的目标是通过连续的X射线结晶学和冷冻电子来完成这些图像。 随后对处于不活跃和活跃状态的代表进行知情的生化分析。 具体目标是：(1)利用时间分辨系列X射线结晶学对生成的中间体进行结构定义 (2)生成更全面的细菌物理结构，包括完整的物理模型。 长度二聚体感光器及其信号输出模块；(3)建立了植物生理信号传递的模型 PFR的结构研究；(4)应用稳态和时间分辨蛋白质表面图谱支持Phy 结构上看到的光转化途径(S)；(5)建立物理与其下游效应器相互作用的模型，以及 (6)了解植物Phy的多样性如何通过特定的异构体实现对温度/时间的感知。 综上所述，这个项目将提供一个基本的框架，以更好地理解结构，变构机制， 以及Phy超级家族的进化。了解微生物和植物如何感知光、温度和时间 将对改善农作物的农业表现、了解微生物 生态系统，控制与医学相关的病原体，并进一步将Phys用作光遗传试剂。

PROJECT SUMMARY Almost all cellular organisms employ an array of photoreceptors to detect their light environment. Arguably the most influential are the phytochromes (Phys), a diverse group essential for plant development and the behavior of many bacterial, fungal, and algal species. By reversible photointerconversion of their bilin (or open-chain tetrapyrrole) chromophores between red light-absorbing Pr and far-red light-absorbing Pfr states, Phys act as photoswitches in various signaling cascades responsive to light intensity, duration, direction, and spectral quality. Moreover, through the thermal reversion of Pfr back to Pr, some Phys sense temperature through enthalpic effects on this reaction, and perceive photoperiod through the nighttime depletion of Pfr. The cumulative effects of this Pr/Pfr interconversion impact numerous physiological processes important to agriculture and the biology of harmful plant and human pathogens. In addition, their unique photochemistries provide invaluable optogenetic tools, including novel fluorophores for tissue imaging, and engineered photoswitches for regulating cellular events with remarkable temporal and spatial precision. Recently, we made great strides in understanding how Phys signal, with emerging structures suggesting that microbial and plant Phys use two distinct output modalities. Both start with light-triggered isomerization of the bilin, which drives a - stranded to -helical rearrangement of a hairpin loop that links the signature PHY and GAF domains. While photoactivated microbial Phys then connect torsional strain generated within the dimer to regulate an appended output domain (typically with histidine kinase activity), plant Phys have rearranged their domain organization to create a photosensitive dimeric platform that likely enables reversible binding and eventual degradation of the family of PIF transcriptional repressors. While current models helped illuminate gross changes required for endstate conversion, the intermediates of photoexcitation and ensuing structural changes necessary for creating a signaling-competent Pfr state remain uncertain. The objectives of this proposal are to complete these pictures through continued X-ray crystallographic and cryo-electron microscopic approaches followed by informed biochemical analyses of representatives in their inactive and active states. Specific aims are to: (1) exploit time-resolved serial X-ray crystallography to structurally define the intermediates generated by Phys after photon absorption; (2) generate more comprehensive structures of bacterial Phys, including models of full- length dimeric photoreceptors with their signal output modules; (3) develop a model for how plant Phys signal through structural studies on Pfr; (4) apply steady-state and time-resolved protein surface mapping to support the Phy photoconversion pathway(s) seen structurally; (5) develop models of Phys interacting with their downstream effectors, and (6) appreciate how diversity among plant Phy enables thermal/time perception by specific isoforms. Taken together, this project will provide an essential framework to better appreciate the structure, allosteric mechanisms, and evolution of the Phy superfamily. Understanding how microorganisms and plants sense light, temperature, and time would then have important ramifications for improving the agricultural performance of crop plants, understanding microbial ecosystems, controlling medically-relevant pathogens, and furthering the application of Phys as optogenetic reagents.